Neomycin

Molecular formula: C23H46N6O13 (neomycin B)

Molecular weight: 614.7 (neomycin B)
CAS Registry No.: 1404-04-2 (neomycin),
1406-04-8 (neomycin undecylenate),
1405-10-3 (neomycin sulfate),
1405-12-5 (neomycin palmitate)

SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. 1 mL Plasma + 100 |JLL 30% trichloroacetic acid in water,
vortex, centrifuge at 4 at 3600 g for 30 min. Remove 450 |xL of the supernatant and add
it to 50 |xL buffer, vortex, centrifuge at 4 at 3600 g for 30 min, inject a 5-25 |JLL aliquot
of the supernatant. Urine. Centrifuge urine at 4 at 3600 g for 30 min. Remove a 180 |xL
aliquot and add it to 20 jxL buffer, vortex, inject a 5-25 |JLL aliquot. (Buffer contained 110
mM sodium 1-pentanesulfonate and 70 mM acetic acid.)
HPLCVARIABLES
Guard column: 20 X 4.6 5 |xm Supelguard LC-8-DB (Supelco)
Column: 150 X 4.6 5 |xm Supelcosil LC-8-DB
Mobile phase: MeOH-.buffer 1.5:98.5 (Buffer contained 11 mM sodium 1-pentanesulfonate,
56 mM sodium sulfate, and 7 mM acetic acid.)
Column temperature: 32.5
Injection volume: 5-30
Detector: F ex 340 em 455 following post-column derivatization. The column effluent mixed
with an o-phthalaldehyde reagent (Pierce) and flowed through a reaction coil (PCR 520,
Applied Biosystems) at 33 to the detector.
CHROMATOGRAM
Retention time: 16
Limit of quantitation: 250 ng/mL (plasma); 1000 ng/mL (urine)
KEYWORDS
plasma; cow; pharmacokinetics; post-column reaction
REFERENCE
Shaikh, B.; Jackson, J.; Guyer, G.; Ravis, W.R. Determination of neomycin in plasma and urine by highperformance liquid chromatography. Application to a preliminary pharmacokinetic study.
J.Chromatogr., 1991, 571, 189-198

SAMPLE
Matrix: bulk
Sample preparation: Dissolve 20 mg neomycin sulfate powder in 100 mL 100 mM pH 8.0
sodium phosphate buffer. Remove a 10 mL aliquot and add it to 10 mL 40 mg/mL 2naphthalenesulfonyl chloride in MeCN (prepare fresh daily), shake briefly, heat at 100105 for 10 min, cool to room temperature, add 15 mL IS solution, shake vigorously for
10 min, centrifuge at <300 g for 3-5 min, inject a 50 JJLL aliquot of the lower organic layer.
(Prepare IS solution by dissolving 2 mg prednisolone in a small amount of THF, make up
to 100 mL with chloroform.)
HPLC VARIABLES
Column: 125 X 4.6 5 jjtm P-E HS-5 silica (Perkin-Elmer)

Mobile phase: Chloroform: MeOH: acetic acid 95:2.3:2.5

Flow rate: 1.7
Injection volume: 50
Detector: UV 254
CHROMATOGRAM
Retention time: 6.2 (neomycin B), 8.3 (neomycin C)
Internal standard: prednisolone (12.3)
OTHER SUBSTANCES
Simultaneous: impurities, neamine
KEYWORDS
derivatization; normal phase
REFERENCE
Tsuji, K.; Jenkins, K.M. Derivatization of primary amines by 2-naphthalenesulfonyl chloride for highperformance liquid chromatographic assay of neomycin sulfate. J.Chromatogr., 1986, 369, 105-115

SAMPLE
Matrix: formulations
Sample preparation: 5 g Ointment + 3 mL MeOH, heat at 55 for 5 min, vortex twice for
20 s, centrifuge at 2000 g for 2 min, discard the supernatant, repeat the MeOH wash
twice more, add 30 mL chloroform, heat at 55, vortex for 15 s, add 10 mL MeOH: water
20:80, shake vigorously for 20 min, centrifuge at 1000 g for 3 min, remove the upper
aqueous layer, repeat the MeOH/water extraction twice more. Combine the aqueous layers and make up to 50 mL with 20 mM pH 9.0 borate buffer. Remove a 10 mL aliquot
and add it to 15 mL 150 mM 2,4-dinitrofluorobenzene in MeOH, heat at 100 for 45 min,
cool, make up to 250 mL with mobile phase, inject an aliquot of the yellow organic layer.
HPLCVARIABLES
Column: 250 X 4.6 5 |mm LiChrosorb silica SI-100
Mobile phase: THF: chloroform: water: glacial acetic acid 39.2:59.8:0.8:0.2
Flow rate: 1
Injection volume: 40
Detector: UV 254
CHROMATOGRAM
Retention time: 4 (neomycin C), 12 (neomycin B)
OTHER SUBSTANCES
Simultaneous: hydrocortisone acetate
Noninterfering: cortisone acetate, fluorometholone, methylprednisolone, prednisolone
acetate
KEYWORDS
normal phase; ointment; derivatization
REFERENCE
Binns, R.B.; Tsuji, K. High-performance liquid chromatographic analysis of neomycin in petrolatumbased ointments and in veterinary formulations. J.Pharm.ScL, 1984, 73, 69-72

SAMPLE
Matrix: formulations

Sample preparation: Powder. Dissolve 50 mg powder in 25 mL 20 mM pH 9.0 borate

buffer, remove a 5 mL aliquot and add it to 15 mL 150 mM 2,4-dinitrofluorobenzene in
MeOH (prepare fresh daily), heat at 100 for 45 min, cool, make up to 250 mL with mobile
phase, discard the upper aqueous phase, inject a 20 JJLL aliquot of the lower organic phase.
Ointment. Weigh out 5 g ointment, add 25 mL chloroform, heat at 60 until the ointment
dissolved, shake vigorously for 15 min, centrifuge at 4000 g for 15 min, discard the chloroform, add 25 mL chloroform to the solid, centrifuge, discard the chloroform, add 10 mL
20 mM pH 9.0 borate buffer and 15 mL n-heptane to the solid, shake vigorously for 10
min, remove a 5 mL aliquot of the aqueous phase and add it to 15 mL 150 mM 2,4dinitrofluorobenzene in MeOH (prepare fresh daily), heat at 100 for 45 min, cool, make
up to 250 mL with mobile phase, discard the upper aqueous phase, inject a 20 |JIL aliquot
of the lower organic phase.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm LiChrosorb SI-100
Mobile phase: Chloroform: THF: water 60:39.2:0.8
Flow rate: 1
Injection volume: 20
Detector: UV 350
CHROMATOGRAM
Retention time: 10 (neomycin C), 14 (neomycin B)
Limit of detection: 1 ng
KEYWORDS
powder; ointment; normal phase; derivatization
REFERENCE
Tsuji, K.; Goetz, J.F.; VanMeter, W.; Gusciora, K.A. Normal-phase high-performance liquid chromatographic determination of neomycin sulfate derivatized with l-fluoro-2,4-dinitrobenzene. J.Chromatogr., 1979, 175, 141-152

SAMPLE
Matrix: milk
Sample preparation: Prepare a SPE column as follows. Shake Amberlite CG 50 resin in
buffer, equilibrate for 2 h, fill a plugged Pasteur pipette to a height of 35 mm with the
slurry, wash with water until the eluent is neutral. 40 mL Milk + 2 g NaCl, shake well,
add a 10 mL aliquot to the SPE column, wash with 8 mL water, add 600 |xL reagent, let
stand for 2 min, elute with 3 mL MeOH: buffer 80:20, make up the eluate to 4 mL with
MeOH, store at -8 to -10, after 15 min inject a 10-20 |xL aliquot. (Prepare buffer by
dissolving 76 g potassium tetraborate in 400 mL water, adjusting pH to 11 with KOH,
and making up to 500 mL with water. Prepare reagent by dissolving 100 mg o-phthalaldehyde in 10 mL MeOH and adding 200 |xL 2-mercaptoethanol and 10 mL borate buffer.
Prepare borate buffer by dissolving 3.1g boric acid in 100 mL water and adjusting the
pH to 10.5 with 50% KOH, make up to 125 mL with water.)
HPLCVARIABLES
Column: 150 X 4.6 5 |xm HISEP (Supelco)
Mobile phase: Gradient. A was 2 g/L tripotassium EDTA in MeOH: water 70:30. B was
MeOH. A:B 100:0 to 40:60 over 15 min (LDC concave curve 4). (This curve holds the
initial conditions for about 6 min.)
Flow rate: 1.7
Injection volume: 10-20
Detector: F ex 340 em KV418 filter
CHROMATOGRAM
Retention time: 8.2, 19.8
Limit of detection: 50 ppb

KEYWORDS
cow; derivatization; SPE
REFERENCE
Agarwal, V.K. High performance liquid chromatographic determination of neomycin in milk using a
HISEP column. J.Liq.Chromatogr., 1990, 13, 2475-2487

SAMPLE
Matrix: milk
Sample preparation: 1 mL Skim milk + 100 |xL 20% trichloroacetic acid, vortex, centrifuge at 4 at 4000 rpm for 30 min. Remove a 180 uX aliquot and add it to 20 |JLL 100 mM
sodium 1-pentanesulfonate containing 70 mM acetic acid, vortex, inject a 25 ^xL aliquot.
HPLCVARIABLES
Guard column: 20 X 4.6 5 jxm Supelguard LC-8-DB
Column: 150 X 4.6 5 fxm Supelcosil LC-8-DB
Mobile phase: MeOH: buffer 1.5:98.5 (Buffer contained 10 mM pentanesulfonic acid, 56
mM sodium sulfate, and 7 mM acetic acid.)
Column temperature: 32.5
Injection volume: 25
Detector: F ex 340 em 455 following post-column reaction. The column effluent mixed with
o-phthalaldehyde solution (Pierce) and flowed through a reaction coil at 33 to the detector.
CHROMATOGRAM
Retention time: 20
Limit of detection: 150 ng/mL
KEYWORDS
cow; derivatization; post-column reaction
REFERENCE
Shaikh, B.; Jackson, J. Determination of neomycin in milk by reversed phase ion-pairing liquid chromatography. J.LJg.Chromatogr., 1989, 12, 1497-1515

SAMPLE
Matrix: perilymph
Sample preparation: Lyophilize 6 jxL perilymph, reconstitute with 90 JJLL pyridine, add
10 |xL benzoyl chloride, heat at 80 for 30 min, evaporate to dryness under a stream of
nitrogen, add 1 mL MeOH, heat at 80 for 10 min, add 50 mg solid sodium carbonate,
add 1 mL MeOH saturated with sodium carbonate, wash 3 times with 2 mL portions of
n-hexane, add 1 mL water, remove any hexane which separates, extract with 3 mL chloroform. Wash the chloroform layer 3 times with 1 mL portions of MeOH: water 50:50,
evaporate the chloroform layer to dryness, reconstitute with 15 |xL chloroform, inject a 5
|xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5-6 jxm Zorbax SIL
Mobile phase: n-Hexane: THF 50:50
Flow rate: 2
Injection volume: 5
Detector: UV 230
CHROMATOGRAM
Retention time: 8.11, 9.28, 10.08
Limit of detection: 10 ng

OTHER SUBSTANCES

Simultaneous: dihydrostreptomycin, kanamycin, streptomycin

KEYWORDS

derivatization; normal phase; guinea pig

REFERENCE
Harada, T.; Iwamori, M.; Nagai, Y.; Nomura, Y. Analysis of aminoglycoside antibiotics as benzoyl derivatives by high-performance liquid chromatography and its application to the quantitation of neomycin in the perilymph. J.Chromatogr., 1985, 337, 187-193

SAMPLE

Matrix: solutions
Sample preparation: Inject a 20 fxL aliquot of a solution in mobile phase.
HPLCVARIABLES

Column: 150 X 4.6 5 |xm Supelcosil LC-8

Mobile phase: THF:buffer 3:97 (Buffer was 7 mM acetic acid containing 5.6 mM sodium
sulfate and 10 mM sodium pentanesulfonate.)
Flow rate: 1.75
Injection volume: 20
Detector: F ex 365 em 418 following post-column reaction. The column effluent mixed with
the reagent pumped at 0.4 mL/min and the mixture flowed through a 50 X 4.6 column
packed with 75 |xm glass beads and a 3.05 m X 0.5 mm knitted PTFE coil at 40 to the
detector. (The reagent was 400 mM boric acid containing 380 mM KOH, 6 mL/L 40% Brij35, 4 mL/L mercaptoethanol, and 0.8 g/L orthophthalaldehyde.)
CHROMATOGRAM

Retention time: 5
KEYWORDS

post-column reaction
REFERENCE
Supelco Chromatography Products, Supelco, Inc., Bellefonte PA, 1996, p. A29

SAMPLE
Matrix: solutions
HPLCVARIABLES

Column: 150 X 4.5 5 jxm Ultrasphere octyl

Mobile phase: MeCN: triethylamine: 1.65% glacial acetic acid 505:0.65:495, pH 4.35
Column temperature: 30
Flow rate: 1
Injection volume: 20
Detector: UV 280
CHROMATOGRAM

Retention time: 1.99

Internal standard: naproxen (3.89)
OTHER SUBSTANCES

Simultaneous: bacitracin, cortisone acetate, diazepam, diclofenac, fluorometholone, flurbiprofen, hydrocortisone acetate, imipramine, indomethacin, ketoprofen, ketorolac tromethamine, levobunolol, meclofenamic acid, prednisolone acetate, proparacaine, salicylic
acid, sulfacetamide, suprofen

Noninterfering: acebutolol, acetaminophen, acetazolamide, alprenolol, apraclonidine,

atenolol, atropine, betamethasone, betaxolol, bupivacaine, caffeine, cyclopentolate, dexamethasone, diphenhydramine, erythromycin, haloperidol, lidocaine, phenylephrine, polymyxin B, procaine, scopolamine, timolol, tropicamide
Interfering: metipranolol, propranolol
KEYWORDS

human; rabbit
REFERENCE
Riegel, M.; Ellis, P.P. High-performance liquid chromatography assay for antiinflammatory agents diclofenac and flurbiprofen in ocular fluids. J.Chromatogr.B, 1994, 654, 140145

SAMPLE

Matrix: solutions
Sample preparation: 50 jxL Buffered reaction mixture + 50 |JLL isopropanol + 50 \xL reagent, heat at 60 for 10 min, centrifuge at 1000 g for 2 min, immediately inject a 50 (xL
aliquot of the supernatant. (Reagent was 80 mM o-phthalaldehyde and 250 mM thioglycolic acid in 1 M boric acid, pH adjusted to 10.4 with 40% KOH.)
HPLCVARIABLES

Column: 100 X 5 Hypersil ODS

Mobile phase: MeOH: water: acetic acid 63.75:31.25:5 containing 5 g/L heptanesulfonic
acid
Flow rate: 2
Injection volume: 50
Detector: UV 330
CHROMATOGRAM

Retention time: 21
KEYWORDS

reaction mixtures; derivatization

REFERENCE
Lovering, A.M.; White, L.O.; Reeves, D.S. Identification of aminoglycoside-acetylating enzymes by highpressure liquid chromatographic determination of their reaction products. Antimicrob.Agents Chemother., 1984,26, 10-12

SAMPLE
Matrix: solutions
HPLCVARIABLES

Column: 300 X 3.9 10 jxm ^Bondapak C18

Mobile phase: MeCN: water 17:83 containing 16 mM sodium 1-hexanesulfonate and 20 mM
Na3PO4, pH 3.5 (Connect a 250 X 4.6 column of Bondapak C18/Corasil or Co:Pell ODS
between pump and injector. Flush column with MeOH: water 50:50 at the end of the day.)
Column temperature: 25
Flow rate: 1.5
Injection volume: 25
Detector: RI
CHROMATOGRAM

Retention time: 8

Next page

OTHER SUBSTANCES
Simultaneous: paromomycin
REFERENCE
Whall, T. J. Determination of streptomycin sulfate and dihydrostreptomycin sulfate by high-performance
liquid ehromatography J.Chromatogr., 1981, 219, 89-100

SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Tissumizer) 1 g Ground tissue + 4 mL buffer at medium speed for 1 min, centrifuge at 3600 g for 20 min, remove the supernatant, re-homogenize pellet in 4 mL buffer for 10 min, centrifuge. Combine the supernatants, heat
in a boiling water bath with occasional mixing for 5 min, centrifuge at 2000 g for 20 min,
remove the supernatant, vortex the precipitate with 2 mL buffer for 30 s, centrifuge at
2000 g for 10 min. Combine the supernatants, acidify to pH 3.5-4 with 50-60 |JLL sulfuric
acid, centrifuge at 2000 g for 10 min, inject an aliquot of the supernatant. (Buffer was
33.46 g K2HPO4 and 1.046 g KH2PO4 in 1 L water, pH 8.0.)
HPLCVARIABLES
Guard column: 10 |jim RP-18
Column: 150 X 4.6 5 \m Supelcosil LC-8-DB
Mobile phase: MeOH: buffer 1.5:98.5 (Buffer was 10 mM sodium 1-pentanesulfonate, 56
mM sodium sulfate, and 7 mM acetic acid.)
Flow rate: 1.5
Detector: F ex 340 em 455 following post-column reaction with derivatization reagent
pumped at 0.9 mL/min. (Derivatization reagent was commercially available (Pierce) or
prepared by adding 2.5 mL 2-mercaptoethanol and 2.5 mL Brij-35 to 850 mg o-phthalaldehyde in 10 mL MeOH, mix until decolorization is complete, add 1 L buffer, filter (0.45
|xm), and refrigerate until used. Buffer was prepared by adjusting pH of 250 mM boric
acid to 9.5 with 5 M KOH.)
CHROMATOGRAM

Retention time: 22
Limit of detection: 3.5 ng
OTHER SUBSTANCES
Extracted: paromomycin
Simultaneous: dihydrostreptomycin, streptomycin
KEYWORDS
kidney; muscle; cow; pig; post-column reaction
REFERENCE
Shaikh, B.; Allen, E.H.; Gridley, J.C. Determination of neomycin in animal tissues, using ion-pair liquid
ehromatography with fluorometric detection. J.Assoc.Off.Anal.Chem., 1985, 68, 29-36