Pentoxifylline

Molecular formula: C13H18N4O3

Molecular weight: 278.3
CAS Registry No.: 6493-05-6

SAMPLE
Matrix: blood
Sample preparation: 100 |JLL Plasma + 100 |xL 20 |Jig/mL tetracaine hydrochloride H- 8
mL dichloromethane, shake for 20 min, centrifuge at 2500 rpm for 20 min. Remove 7 mL
of the organic layer and evaporate it to dryness under nitrogen or at 60. Dissolve residue
in 200 |xL mobile phase, inject a 20 |JLL aliquot.
HPLCVARIABLES
Column: 150 X 6 Shimpack CLS-ODS (Shimadzu)
Mobile phase: MeCN: MeOH: 0.5 mM phosphoric acid 20:12:68

Column temperature: 40
Flow rate: 1.5
Injection volume: 20
Detector: UV 273
CHROMATOGRAM

Internal standard: tetracaine hydrochloride

KEYWORDS

plasma; rat
REFERENCE
Lee, C.K.; Uchida, T.; Kitagawa, K.; Yagi, A.; Kim, N.-S.; Goto, S. Skin permeability of various drugs
with different lipophilicity. J.Pharm.ScL, 1994, 83, 562-565

SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg 1 mL Baker C18 SPE cartridge with 1 mL MeCN
and 1 mL 60 mM pH 5.0 phosphate buffer. 500 |xL Plasma + 150 ng 7-(2'-chloroethyDtheophylline + 100 jxL 200 mM HCl + 400 JJLL 60 mM pH 5.0 phosphate buffer (final
pH 5.3), centrifuge at 10000 g for 3 min. Add the supernatant to the SPE cartridge, wash
twice with 1 mL 60 mM pH 5.0 phosphate buffer, dry, elute three times with 200 |JLL
MeCN. Evaporate the eluate at 37 under a stream of air, reconstitute in 100 JULL mobile
phase, vortex, inject a 20 jxL aliquot.
HPLCVARIABLES
Column: 125 X 4 5 ^m LiChrospher 100 RP-18
Mobile phase: MeCN: dioxan: water 6.5:6.5:87, acidified to pH 3.0 with glacial acetic acid
(0.5% v/v)

Flow rate: 1
Injection volume: 20
Detector: UV 275
CHROMATOGRAM
Retention time: 12.5

Internal standard: 7-(2'-chloroethyl)theophylline (11.2)

Limit of detection: 25 ng/mL
OTHER SUBSTANCES

Extracted: metabolites
Noninterfering: caffeine, theobromine, theophylline
KEYWORDS

plasma; SPE
REFERENCE
Mancinelli, A.; Pace, S.; Marzo, A.; Martelli, E.A.; Passetti, G. Determination of pentoxifylline and its
metabolites in human plasma by high-performance liquid chromatography with solid-phase extraction. J.Chromatogr., 1992, 575, 101-107

SAMPLE

Matrix: blood
Sample preparation: 100 JJLL Plasma + 20 jxL 172 jxM acetophenone + 300 \xL MeOH,
vortex, centrifuge at 1000 g for 5 min, inject a 50 |xL aliquot of the supernatant.
HPLCVARIABLES

Guard column: 5 |xm Hisep (Supelco)

Column: 150 X 4.6 5 |xm Hisep (Supelco)
Mobile phase: MeCN.water 10:90
Injection volume: 50
Detector: UV 280
CHROMATOGRAM

Retention time: 3.3

Internal standard: acetophenone (4.5)
Limit of detection: 3.95 nM
KEYWORDS

plasma; rabbit; pharmacokinetics

REFERENCE
Lockemeyer, M.R.; Smith, CV. Analysis of pentoxifylline in rabbit plasma using a Hisep high-performance liquid chromatography column. J.Chromatogr., 1990, 532, 162167

SAMPLE

Matrix: blood
Sample preparation: 1 mL Plasma + 20 |xL 0.5 mM IS in MeCN: water 22:78 + 100 |xL
3 M HCl + 5 mL dichloromethane: chloroform 1:1, mix on a rotary mixer at 20 rpm for
10 min, centrifuge at 1000 g for 10 min. Remove the organic layer and evaporate it to
dryness under a stream of nitrogen at room temperature. Dissolve the residue in 200 jxL
mobile phase, vortex, centrifuge at 1000 g for 1 min, inject a 20 |xL aliquot.
HPLCVARIABLES

Column: 150 X 4.6 5 |xm Zorbax ODS

Mobile phase: MeCN: water: acetic acid 22:77.9:0.1
Flow rate: 0.75
Injection volume: 50
Detector: UV 274
CHROMATOGRAM

Retention time: 8

Internal standard: l-(6'-oxoheptyl)-3-dimethylxanthine (15)

Limit of detection: 53 nmol/L
OTHER SUBSTANCES

Extracted: metabolites
KEYWORDS

plasma; also urine using SPE

REFERENCE
Lambert, W.E.; Yousouf, M.A.; Van Liedekerke, B.M.; De Roose, J.E.; De Leenheer, A.R Simultaneous
determination of pentoxifylline and three metabolites in biological fluids by liquid chromatography.
Clin.Chem., 1989, 35, 298-301

SAMPLE

Matrix: blood
Sample preparation: Condition a 100 mg Baker cyanopropyl SPE cartridge with 2 mL
MeOH and 2 mL water. 1 mL Plasma + 100 |xL 5 |xg/mL IS in water + 2 mL MeCN,
mix, centrifuge at 600 g for 20 min. Remove the supernatant and evaporate it to dryness
under a stream of nitrogen at 40. Reconstitute the residue in 1 mL water, add it to the
SPE cartridge, wash with 200 JJLL water, elute with 1 mL MeCN. Evaporate the eluate to
dryness under a stream of nitrogen at 45, reconstitute in 1 mL mobile phase, inject a 20
|JLL aliquot.
HPLCVARIABLES

Guard column: 30 X 4 10 \xm LiChrosorb cyanopropyl

Column: 125 X 4 5 |xm LiChrosorb cyanopropyl
Mobile phase: MeCN.water 1:99
Flow rate: 1
Injection volume: 20
Detector: UV 280
CHROMATOGRAM

Retention time: 8
Internal standard: l-(6-oxoheptyl)-3,7-dimethylxanthine (9.5)
Limit of detection: 10 ng/mL
OTHER SUBSTANCES

Extracted: metabolites
Simultaneous: caffeine, chlorthalidone, diclofenac, estriol, furosemide, phenacetin, phenobarbital, theobromine, theophylline, triamterene
Noninterfering: albuterol, amiloride, atenolol, mexiletine, tiapride
KEYWORDS

plasma; SPE
REFERENCE
Musch, G.; Hamoir, T.; Massart, D.L. Determination of pentoxifylline and its 5-hydroxy metabolite in
human plasma by solid-phase extraction and high-performance liquid chromatography with ultraviolet detection. J.Chromatogr., 1989, 495, 215-226

SAMPLE

Matrix: blood
Sample preparation: 250 JULL Whole blood + 4 mL MeCN, vortex for 30 s, centrifuge at
3000 g for 10 min. Remove a 3.7 mL aliquot of the supernatant and add it to 35 |xL 2
|jig/mL IS in MeOH, vortex for 10 s, filter (0.45 |xm PTFE), evaporate the nitrate to dry-

ness under a stream of nitrogen at 47, reconstitute the residue in 2 mL hexane, vortex
for 15 s, add 300 |xL water, vortex for 30 s, centrifuge at 1250 g for 15 min, inject a 250
(xL aliquot of the aqueous phase.
HPLCVARIABLES

Column: 250 X 4.6 5 ixm LC-8-DB (Supelco)

Mobile phase: MeCN: water 24:76
Flow rate: 1
Injection volume: 250
Detector: UV 280
CHROMATOGRAM

Retention time: 6.4

Internal standard: 3-isobutyl-l-methylxanthine (7.6)
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
Simultaneous: antipyrine, caffeine, 8-chlorotheophylline, dimethyluric acid, dyphylline,
hypoxanthine, methyluric acid, methylxanthine, pentifylline, phenacetin, 8-phenyltheophylline, theobromine, uric acid, xanthine
Interfering: pentobarbital
KEYWORDS

whole blood; human; rat

REFERENCE
Grasela, DJVL; Rocci, M.L., Jr. High-performance liquid chromatographic analysis of pentoxifylline and
l-(5'-hydroxyhexyl)-3,7-dimethylxanthinein whole blood. J.Chromatogr., 1987, 419, 368-374

SAMPLE

Matrix: blood
Sample preparation: 250 |xL Plasma + 20 |xL 20 |xg/mL phenacetin in mobile phase,
vortex for 15 s, add 2.5 mL MeCN, vortex for 15 s, centrifuge at 1980 g for 10 min. Remove
the supernatant and filter (PTFE) it, evaporate the nitrate to dryness under a stream of
nitrogen at 70, reconstitute the residue in 125 |xL water, vortex, inject a 100 |xL aliquot.
HPLCVARIABLES

Column: 5 |xm jxBondapak C18

Mobile phase: MeCN: water 24:76
Flow rate: 1
Injection volume: 100
Detector: UV 280
CHROMATOGRAM

Retention time: 8.0

Internal standard: phenacetin (14.7)
Limit of quantitation: 25 ng/mL
OTHER SUBSTANCES

Extracted: metabolites
Simultaneous: aminopyrine, aspirin, caffeine, carbidopa, p-hydroxyethyltheophylline, Phydroxypropyltheophylline, methicillin, methyldopa, nafcillin, nifedipine, penicillamine,
penicillin V, penicillin G, phthalic acid, probenecid, salicylic acid, theobromine,
theophylline

KEYWORDS
plasma; rat; pharmacokinetics
REFERENCE
Luke, D.R.; Rocci, M.L., Jr. Determination of pentoxifylline and a major metabolite, 3,7-dimethyl-l-(5'hydroxyhexyl)xanthine, by high-performance liquid chromatography J.Chromatogr., 1986, 374,
191-195

SAMPLE
Matrix: blood
Sample preparation: Inject a 500 |xL aliquot of plasma onto column A and elute to waste
with mobile phase A, after 4.5 min backflush the contents of column A onto column B
with mobile phase B, monitor the effluent from column B.
HPLCVARIABLES
Column: A 5 X 4.6 25-40 fxm Lichroprep RP 2; B 60 X 4.6 7 jim Nucleosil phenyl
Mobile phase: A 5 mM pH 7.1 Na2HPO4; B MeCN:5 mM pH 6.7 Na2HPO4 23:77
Column temperature: 45
Flow rate: A 1.8; B 0.5
Injection volume: 500
Detector: UV 273
CHROMATOGRAM
Retention time: 5.1
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEYWORDS
plasma; column-switching
REFERENCE
von Stetten, O.; Arnold, P.; Aumann, M.; Guserle, R. Direct measurement of pentoxifylline and its hydroxymetabolite from plasma using HPLC with column switching technique. Chromatographia,
1984, 19, 415-417

SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 jxL aliquot.
HPLCVARIABLES
Column: 150 X 6 5 |xm 3-aminopropylsilyl silica gel with the amino group derivatized with
1,8-naphthalic anhydride (Bunseki Kagaku 1993, 42, 817)
Mobile phase: MeOH: buffer 50:50 (Prepare buffer by dissolving 6.183 g boric acid and
1.461 g NaCl in 500 mL water, adjust pH to 6.4 with sodium borate solution.)
Column temperature: 30
Flow rate: 1
Injection volume: 20
Detector: UV 270
CHROMATOGRAM
Retention time: 20
Internal standard: l,3-dimethyl-7-(2-hydroxyethyl)xanthine (12)

OTHER SUBSTANCES
Simultaneous: caffeine, hypoxanthine, propentofylline, theobromine, theophylline, uric
acid, xanthine
REFERENCE
Nakashima, K.; Inoue, K.; Mayahara, K.; Kuroda, N.; Hamachi, Y; Akiyama, S. Use of 3-(l,8-naphthalimido)propyl-modified silyl silica gel as a stationary phase for the high-performance liquid chromatographic separation of purine derivatives. J.Chromatogr.A, 1996, 722, 107-113

SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 ^m Supelcosil LC-DP (A) or 250 X 4 5 fim LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer
was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust
pH to 3.4 with dilute phosphoric acid, make up to 1 L.)
Flow rate: 0.6
Injection volume: 25
Detector: UV 229
CHROMATOGRAM
Retention time: 5.01 (A), 4.15 (B)
OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine,
albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam,
bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine,
chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine,
chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal,
diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine,
fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac,
labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol,
mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol,
metformin, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine,
methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol,
metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, perphenazine,
pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone,
phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone,
salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin,
trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone

Next page

KEYWORDS

some details of plasma extraction

REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology.
J.ChromatogrA, 1995, 692, 103-119

SAMPLE

Matrix: urine
Sample preparation: 1 mL Urine 100 fxL 1 mg/mL IS in 10 mM NaOH, mix, add 6 mL
dichloromethane, add 1 mL 1 M HCl, extract using a mechanical rotary inversion mixer
at 20 rpm for 15 min, centrifuge at 2000 g for 5 min. Remove the organic phase and
evaporate it to dryness under a stream of nitrogen at 40. Reconstitute the residue in 2
mL mobile phase, inject an aliquot.
HPLC VARIABLES

Column: 150 X 3 5 |xm Spherisorb-ODS 1

Mobile phase: MeOH: 20 mM orthophosphoric acid 1:2.5, adjusted to pH 4 with 6 M NaOH
Flow rate: 1
Detector: UV 274
CHROMATOGRAM

Retention time: 18
Internal standard: H3'-carboxypropyl)-3-methyl-7-propylxanthme(11.6)
OTHER SUBSTANCES

Extracted: metabolites
KEYWORDS

pentoxifylline not usually seen in urine

REFERENCE
Bryce, T.A.; Burrows, J.L.; Jolley, K.W. Determination of l-(3'-carboxypropyl)-3,7-dimethylxanthineand
l-(4'-carboxybutyl)-3,7-dimethylxanthine, two major metabolites of oxpentifylline, in urine by highperformance liquid chromatography. J.Chromatogr., 1985, 344, 397-402

ANNOTATED BIBLIOGRAPHY
Ostrovska, V.; Pechova, A.; Kovacova, D.; Svobodova, X.; Kusala, S. [Determination of pentoxifylline and
its major metabolites in human plasma using HPLC]. Cesk.Farm., 1990, 39, 158-160
Garnier-Moiroux, A.; Poirier, J.M.; Cheymol, G.; Jaillon, P. High-performance liquid chromatographic
determination of pentoxifylline and its hydroxy metabolite in human plasma. J.Chromatogr., 1987,
416, 183-188
Rieck, W.; Platt, D. Determination of 3,7-dimethyl-l-(5-oxohexyl)-xanthine (pentoxifylline) and its 3,7dimethyl-l-(5-hydroxyhexyl)-xanthine metabolite in the plasma of patients with multiple diseases
using high-performance liquid chromatography. J.Chromatogr., 1984, 305, 419-427
Smith, R.V; Yang, S.K.; Davis, PJ.; Bauza, M.T. Determination of pentoxifylline and its major metabolites in microbial extracts by thin-layer and high-performance liquid chromatography.
J.Chromatogr., 1983, 281, 281-287
Chivers, D.A.; Birkett, D.J.; Miners, J.O. Simultaneous determination of pentoxifylline and its hydroxy
metabolite in plasma by high-performance liquid chromatography. J.Chromatogr, 1981, 225, 2 6 1 265