Vol 4 2000.pdf

HPLC METHODS FOR

PHARMACEUTICAL ANALYSIS

Volum es 2

George Lunn

A WILEY-INTERSCIENCE PUBLICATION

JOHN WILEY & SONS, INC.

New York / Chichester / Weinheim / Brisbane / Singapore / Toronto

Paclitaxel

Molecular formula: C 47 H 51 NOi 4

Molecular weight: 853.92

CAS Registry No.: 33069-62-4

Merck Index: 7117

SAMPLE

Matrix: blood

Sample preparation: Add 100 \xL 10 jig/mL docetaxel in MeOH:water 50:50 and 5 mL MeCN:

n-butyl chloride 20:80 to 1 mL plasma, vortex for 5 min, centrifuge at 4000 g for 5 min, evap-

orate the organic layer to dryness under a stream of nitrogen at 60°, reconstitute the residue

with MeOH:water 50:50 with sonication for 1 min, inject a 100 jxL aliquot.

HPLCVARIABLES

Column: 150 X 4.6 5 jxm Inertsil ODS-80A (GL Science, Japan)

Mobile phase: MeOH:THF:water:ammonium hydroxide 60:2.5:37.5:0.1, pH adjusted to 6.0 with

formic acid

Column temperature: 60

Flow rate: 1

Injection volume: 100

Detector: UV 230

CHROMATOGRAM

Retention time: 7.5

Internal standard: docetaxel (8.5)

Limit of quantitation: 10 ng/mL

OTHER SUBSTANCES

Noninterfering: acetaminophen, alizapride, codeine, dexamethasone, domperidone, lorazepam,

metoclopramide, morphine, ranitidine

Interfering: paroxetine

KEYWORDS

pharmacokinetics; plasma

REFERENCE

Sparreboom,A.; de Bruijn,R; Nooter,K; Loos,W.J.; Stoter,G.; Verweij,J. Determination of paclitaxel in human

plasma using single solvent extraction prior to isocratic reversed-phase high-performance liquid chroma-

tography with ultraviolet detection, J.Chromatogr.B, 1998, 705, 159-164.

SAMPLE

Matrix: blood, urine

Sample preparation: Condition a 1 mL Baker Bond cyanopropyl SPE cartridge with 1 mL

MeCN, 1 mL mobile phase, and 1 mL 50 mM KH 2 PO 4 . Free paclitaxel. Add 500 |JLL plasma or

urine to the SPE cartridge, wash with 1 mL water, 1 mL MeCN:water 30:70, and 1 mL 50 mM

KH 2 PO 4 , elute with two 300 |xL portions of mobile phase, inject a 200 |xL aliquot of the eluate.

Total paclitaxel. To 500 |xL plasma or urine add 500 |xL MeOH: 100 mM KH 2 PO 4 50:50 (pH

7.5), hydrolyze at 22° for 20 h, stop the hydrolysis by adding an equal volume of 500 mM

KH 2 PO 4 . Add the hydrolyzed sample to the SPE cartridge, wash with 1 mL water, 1 mL MeCN:

water 30:70, and 1 mL 50 mM KH 2 PO 4 , elute with two 300 (xL portions of mobile phase, inject

a 200 jxL aliquot of the eluate.

HPLC VARIABLES

Guard column: 4 X 4 4 (xm LiChrospher 100 RP-8e

Column: 250 X 4 4 |xm Superspher 60 RP-8e

Mobile phase: MeCN:175 mM pH 4.6 KH 2 PO 4 buffer 55:45

Flow rate: 0.45

Injection volume: 200

Detector: UV 229

CHROMATOGRAM

Retention time: 14

OTHER SUBSTANCES

Extracted: degradation products

KEYWORDS

dog; plasma; SPE

REFERENCE

Fraier,D.; Cenacchi,V.; Frigerio,E. Determination of a new polymer-bound paclitaxel derivative (PNU 166945),

free paclitaxel and 7-epipaclitaxel in dog plasma and urine by reversed-phase high-performance liquid

chromatography with UV detection, J.ChromatogrA, 1998, 797, 295-303.

SAMPLE

Matrix: bulk, formulations

Sample preparation: Bulk. Dilute sample with MeOH containing 0.1% acetic acid. Injections.

Dilute sample with MeCN:water:acetic acid 70:30:0.1. Method 1. Column A, gradient A. Method

2. Column B, gradient B. Method 3. Column A, gradient C. Method 4. Column B, gradient D.

Methods 1 and 2 are for bulk drug potency determination. Methods 3 and 4 are for the deter-

mination of the degradation profile for bulk and injections, potency and content uniformity of

the injections and chromatographic purity profile of the bulk drug.

HPLC VARIABLES

Column: 250 X 4.6 5 |xm Curosil PFP (Phenomenex) (A), 250 X 4.6 5 jxm Whatman TAC-I (PFP)

(Whatman) (B)

Mobile phase: Gradient A. MeCN:water from 40:60 by linear gradient at 0.444%/min until the

paclitaxel peak elutes, return rapidly to initial composition and equilibrate. Gradient B. MeCN:

water 38:62 for 12 min, by linear gradient at 4%/min until paclitaxel peak elutes, return to

initial composition and equilibrate. Gradient C. MeCN:water from 40:60 to 60:40 over 45 min,

to 80:20 over 5 min, hold for 5 min, return to initial composition and equilibrate. Gradient D.

MeCN:water 38:62 for 12 min, to 70:30 over 8 min, hold for 15 min, return to initial composition

and equilibrate.

Flow rate: 1 (column A), 1.5 (column B)

Injection volume: 15

Detector: UV 230

CHROMATOGRAM

Retention time: 24 (Method 1), 22.5 (Method 3), 17 (Method 4)

Limit of detection: 370 ng/mL (method 1), 310 ng/mL (method 2)

Limit of quantitation: 1.11 |jig/mL (method 1), 930 ng/mL (method 2)

OTHER SUBSTANCES

Simultaneous: 13-acetyl-9-dihydrobaccatin, baccatin III, cephalomannine, 10-deacetyl baccatin

III, N-debenzoyl-N-phenylacetyl taxol, 10-deacetyl-7-epi-taxol, 10-deacetyl taxol, 10-deacetyl-

7-xylosyl taxol, 10-deacetyl-7-xylosyl taxol B, 10-deacetyl-7-xylosyl taxol C, 7-epi-taxol, taxinine

M, taxol C, 7-xylosyl taxol

Noninterfering: degradation products, Cremophor EL

KEYWORDS

injections

REFERENCE

Shao,L.K; Locke,D.C. Determination of paclitaxel and related taxanes in bulk drug and injectable dosage forms

by reversed phase liquid chromatography, Anal.Chem., 1997, 69, 2008-2016.

SAMPLE

Matrix: cell culture

Sample preparation: Pulverize dry callus cell culture, pass through 40-mesh sieve. Extract

ultrasonically with MeOH:dichloromethane 10:1 for 30 min. Evaporate extract to dryness, dis-

solve residue in MeOH. Add an aliquot to a Sep-Pak C18 SPE cartridge, wash with water,

wash with MeOH:water 30:70, elute with MeOH:water 85:15. Evaporate the eluate to dryness

and redissolve in a minimum amount of MeOH, inject a 10 (xL aliquot.

HPLC VARIABLES

Column: 250 X 4.6 5 |xm Spherisorb C18

Mobile phase: MeCN:MeOH:water 35:25:45

Flow rate: 1.0

Injection volume: 10

Detector: UV 227

CHROMATOGRAM

Retention time: 27

Limit of quantitation: 11.5 ng

OTHER SUBSTANCES

Extracted: baccatin, cephalomannine, 10-deacetyltaxol, 10-deacetylcephalomannine, baccatin,

10-deacetylbaccatin

KEYWORDS

SPE

REFERENCE

Wu,Y.; Zhu,W. High performance liquid chromatographic determination of taxol and related taxanes from Taxus

callus cultures, J.Liq.Chromatogr., 1997, 20, 3147-3154.

SAMPLE

Matrix: formulations

Sample preparation: Add paclitaxel injection to 0.9% NaCl injection or 5% dextrose injection

to make a paclitaxel concentration of 0.3 or 1.2 mg/mL, mix thoroughly. Inject an aliquot.

HPLC VARIABLES

Column: 250 X 4.6 5 ^m Lichrosphere RP18

Mobile phase: MeOH:water 90:10

Column temperature: 28

Flow rate: 1.3

Injection volume: 500

Detector: UV 273

CHROMATOGRAM

Retention time: 2.1

OTHER SUBSTANCES

Simultaneous: polyoxyethylated castor oil, diethylhexyl phthalate

KEYWORDS

injections

REFERENCE

Mazzo,D.J.; Nguyen-Huu,J.-J.; Pagniez,S.; Denis,R Compatibility of docetaxel and paclitaxel in intravenous

solutions with polyvinyl chloride infusion materials, Am.J.Health-Syst.Pharm., 1997, 54, 566—569.

SAMPLE

Matrix: formulations

Sample preparation: Dilute with mobile phase, inject an aliquot.

HPLCVARIABLES

Column: 250 X 4.6 5 |xm C18

Mobile phase: MeCNiwater 40:60

Flow rate: 2.25

Injection volume: 20

Detector: UV 254

CHROMATOGRAM

Retention time: 4.95

KEYWORDS

stability-indicating; injections; saline

REFERENCE

Mayron,D.; Gennaro,A-R- Stability and compatibility of granisetron hydrochloride in i.v. solutions and oral

liquids and during simulated Y-site injection with selected drugs, Am. J.Health-Syst.Pharm.,

1996, 53, 294-

304.

SAMPLE

Matrix: plant

Sample preparation: Bark. Condition a Supelclean LC-18 SPE cartridge with MeOH and water.

Homogenize (Ystral) 20 g dried bark with 100 mL MeOH. Sonicate the homogenate in a bath

for 10 min, shake at 110 rpm for 30 min, filter through a glass filter. Re-extract residue with

50 mL MeOH, combine two MeOH extracts, evaporate to dryness at 40-45°, reconstitute the

residue with 5 mL MeOH. Add a 500 (JLL aliquot to the SPE cartridge. Needles, clippings. Add

3 g sample to 100 mL chloroform:EtOH 50:50 (Caution! Chloroform is a carcinogen!), sonicate,

filter though a glass filter, evaporate the filtrate to dryness, reconstitute the residue in 5 mL

MeOH. Add a 500 JJLL aliquot to the SPE cartridge. Wash the cartridge twice with 2 mL portions

of water, with 2 mL MeOH:water 20:80, and with 2 mL MeOH:water 50:50. Elute with 2 mL

MeOH, evaporate the fractions to dryness in a speed vac, reconstitute the residue with two

100 |xL aliquots of MeCN, inject 10 |xL aliquot.

HPLC VARIABLES

Guard column: 20 X 4.6 10 |xm Lichrosorb RP-18

Column: 150 X 3.9 4 |xm Novapak Phenyl

Mobile phase: Gradient. A was MeCN:50 mM ammonium acetate 30:70. B was MeCN:50 mM

ammonium acetate 90:10. A:B 100:0, to 66:34 over 30 min, return to initial conditions over 2

min.

Flow rate: 0.8 (UV), 1 (MS)

Injection volume: 10

Detector: UV 227; MS, Finnigan MAT TSQ-70, electrospray, positive ion mode 150 V, sheath

flow MeOH:water:acetic acid 80:20:1, mobile phase split 19:1 before MS

CHROMATOGRAM

Retention time: 22 (UV), 20 (MS)

OTHER SUBSTANCES

Extracted: baccatin III, cephalomannine, 10-DAB, 10-deacetyltaxol, 7-epi-10-deacetyltaxol, 7-

epi-taxol, taxinine M, taxol C, 7-xylosyl-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol B, 7-xylo-

syl-10-deacetyltaxol C, 7-xylosyl-taxol

KEYWORDS

bark; needles; clippings; SPE

REFERENCE

Theodoridis,G.; Laskaris,G.; de Jong,C.R; Hofte,A-J-P«; Verpoorte,R. Determination of paclitaxel and related

diterpenoids in plant extracts by high-performance liquid chromatography with UV detection in high-per-

formance liquid chromatography-mass spectrometry, J.ChromatognA, 1998, 802, 297-305.

SAMPLE

Matrix: plants

Sample preparation: Extract needles with MeOH, concentrate the extract under reduced pres-

sure at <30°, partition concentrate with 0.8 volumes of water and 0.8 volumes of chloroform,

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