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Diclofenac
Molecular formula: C14H11CI2NO2 Molecular weight: 295.1 CAS Registry No.: 15307-79-6 (diclofenac sodium)

SAMPLE Matrix: aqueous humor Sample preparation: 100 |xL Aqueous humor + 500 |xL MeCN + 30 |xL 400 ng/mL (+)naproxen in MeOH, mix mechanically for 90 s, centrifuge at 3000 g for 20 min. Remove the supernatant and dry it under nitrogen at room temperature, dissolve the residue in 50 |JLL mobile phase by swirl-mixing for 1 min, centrifuge at 3000 g for 20 s, reduce volume to 20-30 JJLL, inject an aliquot. HPLCVARIABLES Column: 150 X 4.5 5 |xm 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: 7.14 Internal standard: naproxen (3.89) Limit of detection: 0.3 ng OTHER SUBSTANCES Extracted: flurbiprofen, indomethacin, meclofenamic acid Simultaneous: bacitracin, cortisone acetate, diazepam, fluorometholone, hydrocortisone acetate, imipramine, ketoprofen, ketorolac tromethamine, levobunolol, metipranolol, neomycin, prednisolone acetate, proparacaine, propranolol, 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 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, 140-145

SAMPLE Matrix: blood Sample preparation: 500 |xL Plasma + 1 |xg naproxen + 500 fxL 500 mM HCl, vortex for 1 min, add 5 mL ethyl acetate, extract for 20 min, centrifuge at 2500 rpm for 10 min. Remove the organic layer and evaporate it to dryness, reconstitute the residue in 200 |xL MeCN, inject a 100 JJLL aliquot. HPLCVARIABLES Column: 250 X 5 5 |xm C18 (Machery & Nagel)

Mobile phase: MeCN: water: acetic acid 50:50:0.1 Flow rate: 1 Injection volume: 100 Detector: UV 280 CHROMATOGRAM Internal standard: naproxen Limit of quantitation: 50 ng/mL KEYWORDS plasma; pharmacokinetics REFERENCE
Ramakrishna, S.; Fadnavis, N.W.; Diwan, RV. Comparative pharmacokinetic evaluation of compressed suppositories of diclofenac sodium in humans. Arzneimittelforschung, 1996, 46, 175-177

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 50 jxL 10 |xg/mL flufenamic acid in MeCN + 4 mL MeCN, vortex for 1 min, centrifuge at 2500 rpm for 10 min. Remove the supernatant and evaporate it to dryness under a stream of nitrogen at 45, reconstitute the residue in 200 IxL mobile phase, vortex for 30 s, centrifuge at 11500 rpm for 2 min, inject a 140 JAL aliquot of the supernatant. HPLCVARIABLES Column: 250 X 4.6 5 |xm Supelcosil LC-8 Mobile phase: MeCN: water 50:50 adjusted to pH 3.3 with glacial acetic acid (After 13 min increase flow rate to 2.7 mL/min over 4 min, maintain at 2.7 mL/min for 11 min, return to initial conditions.) Flow rate: 2 Injection volume: 140 Detector: UV 280 CHROMATOGRAM Retention time: 6 Internal standard: flufenamic acid (10) Limit of detection: 25 ng/mL OTHER SUBSTANCES Extracted: nitrofenac (UV 275), metabolites KEYWORDS plasma; rat; pharmacokinetics REFERENCE
Benoni, G.; Terzi, M.; Adami, A.; Grigolini, L.; Del Soldato, R; Cuzzolin, L. Plasma concentrations and pharmacokinetic parameters of nitrofenac using a simple and sensitive HPLC method. J.Pharm.Sci., 1995, 84, 93-95

SAMPLE Matrix: blood Sample preparation: 500 jxL Plasma + 600 |xL 1 M phosphoric acid, vortex for 10 s, add 5 mL 30 ng/mL diphenylamine in hexane: isopropanol 95:5, vortex for 1 min, centrifuge at 1000 g for 10 min. Remove 4 mL of the organic layer and evaporate it to dryness under a stream of air at 40, reconstitute the residue in 150 |xL mobile phase, vortex for 30 s, inject a 25 |xL aliquot.

HPLC VARIABLES

Column: 200 X 4.6 10 |xm Spherisorb ODS Mobile phase: MeOH:buffer 68:32 (Buffer was 6.8 g/L sodium acetate adjusted to pH 4.2 with HCl.) Flow rate: 1.4 Injection volume: 25 Detector: UV 274
CHROMATOGRAM

Retention time: 4.8 Internal standard: diphenylamine (6.4) Limit of detection: 30 ng/mL Limit of quantitation: 100 ng/mL
OTHER SUBSTANCES

Noninterfering: aspirin, chlorphenpyridamine, ciprofloxacin, ibuprofen, lomefloxacin, norfloxacin, ofloxacin

KEYWORDS

plasma; pharmacokinetics
REFERENCE
Li, K.; Zhao, R-L.; Yuan, Y-S.; Tan, L. Determination of diclofenac sodium in human plasma by reversedphase liquid chromatography. J.Liq.Chromatogr., 1995, 18, 2205-2216

SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 1 jxg naproxen + 100 |xL 5% zinc sulfate in water, vortex for 2 min, add 3 mL MeOH, vortex for 2 min, add 440 (JLL buffer, vortex for 1 min, centrifuge at 27 at 2000 g for 10 min, inject a 100 uL aliquot of the supernatant. (Buffer was 100 mM NaH2PO4 containing 10 mM sodium lauryl sulfate, adjust pH to 2.8 with orthophosphoric acid, filter (0.45 |xm).)
HPLCVARIABLES

Column: 250 X 4.6 5 ^m Nucleosil C18 Mobile phase: MeCN: water 35:65 containing 1 mM sodium lauryl sulfate and 10 mM NaH2PO4, pH adjusted to 2.8 with orthophosphoric acid Flow rate: 1.5 Injection volume: 100 Detector: UV 280
CHROMATOGRAM

Retention time: 10.6 Internal standard: naproxen (5.8) Limit of detection: 30 ng/mL
KEYWORDS

plasma; pharmacokinetics
REFERENCE
Mason, J.L.; Hobbs, G.J. A rapid high performance liquid chromatographic assay for the measurement of diclofenac in human plasma. J.Liq.Chromatogr., 1995, 18, 2045-2058

SAMPLE Matrix: blood

Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanol: n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.) HPLCVARIABLES Column: 300 X 3.9 4 jxm NovaPack C18 Mobile phase: MeOH:THF-.buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.) Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 275 CHROMATOGRAM Retention time: 9.51 Limit of detection: <120 ng/mL OTHER SUBSTANCES Extracted: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen, aconitine, ajmaline, albuterol, alimemazine, alminoprofen, alpidem, alprazolam, alprenolol, amisulpride, amodiaquine, amoxapine, aspirin, astemizole, atenolol, benazepril, benperidol, benzocaine, benzoylecgonine, bepridil, betaxolol, bisoprolol, bromazepam, brompheniramine, bumadizone, bupivacaine, buprenorphine, buspirone, caffeine, carbamazepine, carbinoxamine, carpipramine, carteolol, celiprolol, cetirizine, chlorambucil, chlordiazepoxide, chlormezanone, chlorophenacinone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, cibenzoline, cicletanine, clemastine, clobazam, clomipramine, clonazepam, clonidine, clorazepate, clozapine, cocaine, codeine, colchicine, cyamemazine, cyclizine, cycloguanil, cyproheptadine, cytarabine, dacarbazine, daunorubicin, debrisoquine, demexiptiline, desipramine, dextromethorphan, dextromoramide, dextropropoxyphene, diazepam, diazoxide, dihydralazine, diltiazem, diphenhydramine, dipyridamole, disopyramide, dosulepine, doxepin, doxylamine, droperidol, ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentiazac, flecainide, floctafenine, flumazenil, flunitrazepam, fluoxetine, fluphenazine, flurbiprofen, fluvoxamine, glibenclamide, glibornuride, glipizide, glutethimide, haloperidol, histapyrrodine, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketamine, ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, lisinopril, loperamide, loprazolam, loratadine, lorazepam, loxapine, maprotiline, medazepam, medifoxamine, mefenamic acid, mefenidramine, melphalan, meperidine, mephenesin, mephentermine, mepivacaine, metapramine, metformin, methadone, methaqualone, methocarbamol, methotrexate, metipranolol, metoclopramide, metoprolol, mexiletine, mianserine, midazolam, minoxidil, moclobemide, moperone, morphine, nadolol, nalbuphine, nalorphine, naloxone, naltrexone, naproxen, nialamide, nicardipine, nifedipine, niflumic acid, nimodipine, nitrazepam, nitrendipine, nizatidine, nomifensine, omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phencyclidine, phenobarbital, phenol, phenylbutazone, pimozide, pindolol, pipamperone, piroxicam, prazepam, prazosin, prilocaine, procainamide, procarbazine, proguanil, promethazine, propafenone, propranolol, protriptyline, pyrimethamine, quinidine, quinine, quinupramine, ramipril, ranitidine, reserpine, ritodrine, secobarbital, sotalol, strychnine, sulfinpyrazole, sulindac, sulpride, sultopride, suriclone, temazepam, tenoxicam, terbutaline, terfenadine, tetracaine, tetrazepam, thiopental, thioproperazine, thioridazine, tianeptine, tiapride, tiaprofenic acid, ticlopidine, timolol, tofisopam, tolbutamide, toloxatone, trazodone, triazolam, trifluoperazine, trifluperidol, triprolidine, tropatenine, verapamil, viloxazine, vinblastine, vincristine, vindesine, warfarin, yohimbine, zolpidem, zopiclone, zorubicine Interfering: amitriptyline, diclofenac, mefloquine, nortriptyline, tioclomarol, trimipramine

KEYWORDS whole blood; plasma REFERENCE

Tracqui, A.; Kintz, P.; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic Sci., 1995, 40, 254-262

SAMPLE Matrix: blood Sample preparation: 0.5 mL Plasma + 10 (xL 50 |xg/mL mefenamic acid + 50 |xL 85% phosphoric acid, vortex 10 sec, add 3 mL chloroform, vortex 1 min, centrifuge at 6000 rpm for 5 min. Remove organic layer and evaporate it to dryness at 45 under a stream of nitrogen. Vortex residue with 200 jxL mobile phase for 10 s, inject 50 |xL aliquot. HPLC VARIABLES Guard column: 30-40 juim C18 pellicular Column: 150 X 3.9 Novapak C18 Mobile phase: MeCN: water 50:50 adjusted to pH 3.5 with glacial acetic acid Flow rate: 1.5 Injection volume: 50 Detector: UV 278 CHROMATOGRAM Retention time: 3.8 Internal standard: mefenamic acid (6.3) Limit of quantitation: 10 ng/mL KEYWORDS plasma; dog REFERENCE
Mohamed, F.A.; Jun, H.W.; Elfaham, T.H.; Sayed, H.A.; Hafez, E. An improved HPLC procedure for the quantitation of diclofenac in plasma. J.Liq.Chromatogr., 1994, 17, 1065-1088

SAMPLE Matrix: blood Sample preparation: Deproteinize plasma with HCl and extract with dichloromethane. Remove the organic layer and evaporate it to dryness, reconstitute the residue in 100 |xL mobile phase, inject a 40 |xL aliquot. HPLCVARIABLES Column: 150 X 3.6 ODS Hypersil Mobile phase: MeCN: isopropanol: pH 7 sodium acetate buffer .water 21:6:20:53 Flow rate: 0.4 Injection volume: 40 Detector: UV 280 CHROMATOGRAM Limit of detection: 20 ng/mL OTHER SUBSTANCES Noninterfering: ranitidine KEYWORDS plasma; bioequivalence

REFERENCE
Van Gelderen, M.E.M.; Oiling, M.; Barends, D.M.; Meulenbelt, J.; Salomons, P.; Rauws, A.G. The bioavailability of diclofenac from enteric coated products in healthy volunteers with normal and artificially decreased gastric acidity. Biopharm.Drug Dispos., 1994, 15, 775-788

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 200 (xL 1.5 |xg/mL IS in water + 4 mL 2 M phosphoric acid + 6 mL hexane: isopropanol 9:1, shake at 150 oscillations/min for 15 min, centrifuge at 1500 g for 10 min. Remove organic layer and evaporate it to dryness at 37 under a gentle stream of nitrogen. Reconstitute in 250 JXL mobile phase, inject 100 JXL aliquot onto column A, after 2 min switch eluent from column A onto column B, after another 2 min switch column A out of circuit and continue to flush it to waste with mobile phase. Monitor eluent from column B. HPLCVARIABLES Column: A 35 X 4.6 10 |xm Nucleosil C18; B 150 X 4.6 10 \xm Nucleosil C18 Mobile phase: MeCN: MeOH: 22 mM pH 7.1 sodium acetate 23:25:52 (both columns) Flow rate: 1.5 Injection volume: 100 Detector: UV 280 CHROMATOGRAM Retention time: 6.4 Internal standard: CGP-4287 (7.6) Limit of detection: 2.5 ng/mL KEYWORDS plasma; column-switching REFERENCE
Miller, R.B. High-performance liquid chromatographic determination of diclofenac in human plasma using automated column switching. J.Chromatogr., 1993, 616, 283-290

SAMPLE Matrix: blood Sample preparation: 50 |xL Plasma + 50 u-L MeCN, mix, centrifuge at 12000 g for 2 min, inject a 50 |xL aliquot of the supernatant. HPLCVARIABLES Guard column: 15 X 3.2 5 |xm NewGuard ODS Column: 150 X 4 5 |xm Nucleosil C18 Mobile phase: MeCN: buffer 32:68 (Buffer was 40 mL 1 M NaH2PO4 and 40 mL 500 mM Na2HPO4 made up to 680 mL with water, pH 6.6.) Flow rate: 0.7 Injection volume: 50 Detector: F ex 288 em 360 following post-column reaction. The column effluent flowed through a 1.3 m X 1 mm ID PTFE tube irradiated by a UV 254 lamp (Philips TUV 6W, TYP 103314) to the detector. CHROMATOGRAM Retention time: 10 Limit of detection: 6 ng/mL KEYWORDS post-column reaction; plasma; human; rat

REFERENCE
Wiese, B.; Hermansson, J. Bioanalysis of diclofenac as its fluorescent carbazole acetic acid derivative by a post-column photoderivatization high performance liquid chromatographic method. J.Chromatogr., 1991,567, 175-183

SAMPLE

Matrix: blood, CSF Sample preparation: Condition a Baker SPE-Octadecyl (C 18) SPE cartridge with 2 mL MeOH and 1 mL 1 M phosphoric acid. 0.5 mL Plasma or CSF + 2.5 ng (CSF) or 50 ng (plasma) pirprofen, shake, add 1 mL of 1 M phosphoric acid, add to the cartridge, wash twice with 1 mL 1 M phosphoric acid, wash twice with 1 mL water, elute with two 250 |JLL aliquots of MeOH. Evaporate MeOH at room temperature under a stream of nitrogen, dissolve residue in 100 |xL mobile phase, inject 10-20 |JLL.
HPLCVARIABLES

Column: 80 X 4.6 3jxm Perkin-Elmer C8 Mobile phase: MeCN: buffer 35:65 (Buffer was 50 mM sodium acetate adjusted to pH 3.00 with phosphoric acid.) Flow rate: 1.2 Injection volume: 10-20 Detector: E, BAS LC-4B/17AT glassy carbon electrode, Ag/AgCl reference electrode, +0.95 V
CHROMATOGRAM

Retention time: 23 Internal standard: pirprofen (12) Limit of detection: 0.7 ng/mL
OTHER SUBSTANCES

Extracted: metbolites
KEYWORDS

plasma; SPE
REFERENCE
Zecca, L.; Ferrario, P.; Costi, P. Determination of diclofenac and its metabolites in plasma and cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection. J.Chromatogr., 1991, 567, 425-432

SAMPLE

Matrix: blood, exudate Sample preparation: 100 |xL Plasma or exudate + 50 J U L L 50 |xg/mL IS + 250 |xL 900 mM phosphoric acid, vortex, add 2 mL hexane: isopropanol 90:10, rotate for 10 min, centrifuge, freeze. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 45, reconstitute the residue in 120 |xL mobile phase, inject a 100 fxL aliquot.
HPLCVARIABLES

Column: 200 X 4.6 Hypersil ODS Mobile phase: MeOH: MeCN: 1% acetic acid 55:16:29 Flow rate: 1 Injection volume: 100 Detector: UV 282
CHROMATOGRAM

Internal standard: 2-(p-cyclohexen-l'-ylphenyl)propionicacid Limit of detection: 100 ng/mL

KEYWORDS plasma; rat; pharmacokinetics REFERENCE

Stevens, A.J.; Martin, S.W.; Brennan, B.S.; McLachlan, A.; Gifford, L.A.; Rowland, M.; Houston, J.B. Regional drug delivery II: Relationship between drug targeting index and pharmacokinetic parameters for three non-steroidal anti-inflammatory drugs using the rat air pouch model of inflammation. Pharm.Res., 1995, 12, 1987-1996

SAMPLE Matrix: blood, synovial fluid Sample preparation: 0.5 mL Plasma or synovial fluid + 50 JJLL 24 |xg/mL flurbiprofen + 200 JJLL 2 M HCl + 5 mL hexane, tumble 10 min on a rotary mixer, centrifuge at 10 000 g for 5 min. Remove organic layer and evaporate it to dryness under vacuum centrifugation. Reconstitute residue in 150 jxL MeOH + 100 |xL water, vortex mix, inject aliquot. HPLCVARIABLES Guard column: 20 X 2 Perisorb RP18 30-40 \m pellicular Column: 125 X 4.6 5 |xm Spherisorb ODS 1 Mobile phase: MeOH: water 63:37 adjusted to pH 3.3 with phosphoric acid Flow rate: 1 Injection volume: 20 Detector: UV 280 CHROMATOGRAM Retention time: 7.5 Internal standard: flurbiprofen (9) Limit of detection: <100 ng/mL KEYWORDS plasma REFERENCE
Blagbrough, LS.; Daykin, M.M.; Doherty, M.; Pattrick, M.; Shaw, RN. High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man. J.Chromatogr., 1992, 578, 251-257

SAMPLE Matrix: blood, urine Sample preparation: Plasma. 500 jxL Plasma + 50 |xL 20 uM mefenamic acid + 200 |xL 2 M HCl + 5 mL dichloromethane, rotate for 10 min, centrifuge at 5000 rpm for 8 min. Remove 4 mL of the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 250 JJLL plasma mobile phase, inject a 100 JJLL aliquot. Urine. 100 jxL Urine + 100 JJLL 400 p,g/mL ascorbic acid + 100 |xL 5 M NaOH, vortex for 30 s, heat at 75for 1 h, add 50 |xL 20 \xM mefenamic acid, add 500 [xL 2 M HCl, add 5 mL dichloromethane, rotate for 10 min, centrifuge at 5000 rpm for 8 min. Remove 4 mL of the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 500 |xL urine mobile phase, inject a 100 jxL aliquot. HPLCVARIABLES Guard column: 4 x 4 4 |xm Lichrocart C18 (Merck) Column: 50 X 4 4 jjtm Lichrocart C18 (Merck) Mobile phase: MeCN: 100 mM pH 7.4 phosphate buffer: triethylamine 25:75:0.02 (plasma) or 20:80:0 (urine) Flow rate: 1

Injection volume: 100 Detector: UV 282

CHROMATOGRAM

Internal standard: mefenamic acid

OTHER SUBSTANCES

Extracted: metabolites Noninterfering: fluvastatin

KEY WORDS

plasma; pharmacokinetics
REFERENCE
Transon, C; Leemann, T.; Vogt, N.; Dayer, P. In vivo inhibition profile of cytochrome P450TB (CYP2C9) by ()-fluvastatin. Clin.Pharmacol.Ther., 1995, 58, 412-417

SAMPLE

Matrix: bulk, formulations Sample preparation: Powder tablets, shake (ca. 100 mg diclofenac) with 25 mL MeCN: water 25:75 for 30 min, centrifuge at 3500 rpm for 10 min.
HPLCVARIABLES

Column: 250 X 4.6 Chromatography Sciences Co. octadecylsilane bonded phase Mobile phase: MeCN: THF: buffer 300:75:625 (Buffer was 50 mM (NH4)H2PO4 adjusted to pH 5.0 with 50 mM ammonium hydroxide.) Flow rate: 1.5 Injection volume: 10 Detector: UV 229
CHROMATOGRAM

Retention time: 17 Limit of quantitation: 400 ng/mL

KEYWORDS

tablets
REFERENCE
Beaulieu, N.; Lovering, E.G.; Lefrangois, J.; Ong, H. Determination of diclofenac sodium and related compounds in raw materials and formulations. J.Assoc.Off.Anal.Chem., 1990, 73, 698-701

SAMPLE

Matrix: perfusate Sample preparation: 250 |JLL Perfusate + 500 |xL MeCN, vortex for 1 min, centrifuge for 10 min, inject a 150 fxL aliquot of the supernatant.
HPLCVARIABLES

Column: 100 mm long 5 juim Radpak C18 Mobile phase: MeOH: water: triethylamine 70:30:0.2 adjusted to pH 7 with concentrated phosphoric acid Injection volume: 150 Detector: UV 273
CHROMATOGRAM

Limit of quantitation: 100 ng/mL

REFERENCE
Bassily, M.; Ghabrial, H.; Smallwood, R.A.; Morgan, D.J. Determinants of placental drug transfer: Studies in the isolated perfused human placenta. J.Pharm.Sci., 1995, 84, 1054-1060

SAMPLE Matrix: perfusate HPLCVARIABLES Column: 250 X 4.6 Nucleosil 100-5C18 Mobile phase: MeOH: 18.7 mM phosphoric acid 80:20 Flow rate: 1 Detector: UV 282 REFERENCE
Takahashi, K.; Suzuki, T.; Sakano, H.; Mizuno, N. Effect of vehicles on diclofenac permeation across excised rat skin. Biol.Pharm.BulL, 1995, 18, 571-575

SAMPLE Matrix: solutions HPLCVARIABLES Column: 125 X 3 Ecocart LiChrospher 100 RP-18 Mobile phase: Isopropanol.lOO mM KH2PO4:formic acid 54:100:0.1 Flow rate: 0.6 Detector: UV 254 CHROMATOGRAM Retention time: 14.7 Limit of quantitation: 200-500 ng/mL OTHER SUBSTANCES Simultaneous: acemetacin, fiurbiprofen, indomethacin, lonazolac, ketoprofen, naproxen, piroxicam, sulindac, tenoxicam REFERENCE
Baeyens, W.R.G.; Van Der Weken, G.; Van Overbeke, A.; Zhang, Z.D. Preliminary results on the LCseparation of non-steroidal anti-inflammatory agents in conventional and narrow-bore RP set-ups applying columns with different internal diameters, Biomed.Chromatogr., 1995, 9, 261-262

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 5 ^m Supelcosil LC-DP (A) or 250 X 4 5 jxm 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: 8.68 (A), 9.97 (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, diflunisal, diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fiuoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ievorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, 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, pentoxifylline, 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 KEYWORDS some details of plasma extraction REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology. J.Chromatogr.A, 1995, 692, 103-119

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210 OTHER SUBSTANCES Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotro-

pine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triproilidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine REFERENCE
Hill, D. W.; Kind, A. J. Reversed-phase solvent gradient HPLC retention indexes of drugs. J.Anal.ToxicoL, 1994, 18, 233-242

SAMPLE Matrix: urine Sample preparation: 250 JJLL Urine + 50 jxL 12.5 |xg/mL 4'-hydroxy-5-chlorodiclofenac in MeOH + 150 |xL 5 M NaOH, vortex at medium speed for 5-10 s, heat at 70 for 1 h, cool to room temperature, neutralize with 1 M HCl, add 750 |xL buffer, vortex, add 7 mL dichloromethane: isopropanol 95:5, shake horizontally at 180 cycles/min for 10 min, centrifuge at 750 g for 5 min. Remove 5 mL of the organic layer and evaporate it to dryness under a vacuum at 30, reconstitute the residue in 500 |xL mobile phase containing 0.1% ascorbic acid, inject a 20 |xL aliquot. (Buffer was 877 mL 1 M KH2PO4 and 123 mL 1 M Na2HPO4, pH 6.0.)

HPLCVARIABLES Column: 150 mm long 5 |xm ODS (Supelco) Mobile phase: Gradient. MeOH:MeCN:buffer 57.5:0.3:42.2 for 12 min, to 57.5:1.5:41.0 over 2 min, maintain at 57.5:1.5:41.0 for 12 min, re-equilibrate at initial conditions for 4 min. After 12 min increase flow rate to 1.6 mL/min over 2 min, maintain at 1.6 mL/min for 12 min, return to initial conditions. (Buffer was 1.156 g (NH4)H2PO4 in 1 L water adjusted to pH 2.66 with concentrated phosphoric acid.) Flow rate: 1 Injection volume: 20 Detector: UV 270 CHROMATOGRAM Retention time: 21.7 Internal standard: 4'-hydroxy-5-ehlorodiclofenae (18.66) Limit of quantitation: 400 ng/mL OTHER SUBSTANCES Extracted: metabolites KEYWORDS pharmacokinetics REFERENCE Sawchuk, R.J.; Maloney, J.A.; Cartier, L.L.; Rackley, R.J.; Chan, K.K.H.; Lau, H.S.L. Analysis of diclofenac and four of its metabolites in human urine by HPLC. Pharm.Res., 1995, 12, 756-762

ANNOTATED BIBLIOGRAPHY Hanses, A.; Spahn-Langguth, H.; Mutschler, E. A new rapid and sensitive high-performance liquid chromatographic assay for diclofenac in human plasma. Arch.Pharm.fWeinheim)., 1995, 328, 257-260 Fukuyama, T.; Yamaoka, K.; Ohata, Y.; Nakagawa, T. A new analysis method for disposition kinetics of enterohepatic circulation of diclofenac in rats. Drug Metab.Dispos., 1994, 22, 479-485 [rat; plasma; bile; column temp 40] Maitani, Y.; Kugo, M.; Nagai, T. Permeation of diclofenac salts through silicone membrane: A mechanistic study of percutaneous absorption of ionizable drugs. Chem.Pharm.Bull., 1994, 42, 1297-1301 Oberle, R.L.; Das, H.; Wong, S.L.; Chan, K.K.; Sawchuk, R.J. Pharmacokinetics and metabolism of diclofenac sodium in Yucatan miniature pigs. Pharm.Res., 1994, 11, 698-703 [pharmacokinetics; pig; metabolites; plasma; indomethacin (IS); LOQ 50 ng/mL] Reer, O.; Bock, T.K.; Muller, B.W. In vitro corneal permeability of diclofenac sodium in formulations containing cyclodextrins compared to the commercial product Voltaren ophtha. J.Pharm.Sci., 1994, 83, 1345-1349 [perfusate] Zhang, S.Y.; Zou, H.Q.; Zhang, Z.Y.; Peng, W.L.; Liu, L.Q. [High-performance liquid chromatographic method for the determination of diclofenac in serum and its pharmacokinetics in healthy volunteers]. Yao Hsueh Hsueh Pao, 1994, 29, 228-231 Avgerinos, A.; Karidas, T.; Malamataris, S. Extractionless high-performance liquid chromatographic method for the determination of diclofenac in human plasma and urine. J.Chromatogr., 1993, 619, 324-329 [plasma; urine; indomethacin (IS); column temp 40; LOD 200 ng/mL; pharmacokinetics] De Bernardi di Valserra, M.; Feletti, R; Tripodi, A.S.; Contos, S.; Carabelli, A.; Maggi, L.; Germogli, R. Pharmacokinetic studies in healthy volunteers on a new gastroprotective pharmaceutic form of diclofenac. Arzneimittelforschung, 1993, 43, 373-377 [plasma; column temp 30; LOD 20 ng/mL; pharmacokinetics] Schmitz, G.; Lepper, H.; Estler, C-J. High-performance liquid chromatographic method for the routine determination of diclofenac and its hydroxy and methoxy metabolites from in vitro systems. J.Chromatogr., 1993, 620, 158-163 [gradient; cell suspensions; extracted metabolites; LOD 5 ng/mL]

Szasz, G.; Budvari-Barany, Z.; Lore, A.; Radeczky, G.; Shalaby, A. HPLC of antiphlogistic acids on silica dynamically modified with cetylpyridinium chloride. J.Liq.Chromatogr, 1993, 16, 2335-2345 [fenoprofen, ibuprofen, ketoprofen, naproxen, nicotinic acid, nifluminic acid, salicylic acid] Moncrieff, J. Extractionless determination of diclofenac sodium in serum using reversed-phase highperformance liquid chromatography with fluorimetric detection. J.Chromatogr., 1992, 577, 185-189 [fluorescence detection; harmol (IS); serum; column temp 40; LOD 20 ng/mL] Santos, S.R.; Donzella, H.; Bertoline, M.A.; Pereira, M.D.; Omosako, CE.; Porta, V. Simplified micromethod for the HPLC measurement of diclofenac in plasma. Braz.J.Med.Biol.Res., 1992, 25, 125128 Brunner, L.A.; Luders, R.C. An automated method for the determination of diclofenac sodium in human plasma. J.Chromatogr.ScL, 1991, 29, 287-291 [plasma; LOQ 5 ng/mL; LOD 2.5 ng/mL] Sioufi, A.; Richard, J.; Mangoni, P.; Godbillon, J. Determination of diclofenac in plasma using a fully automated analytical system combining liquid-solid extraction with liquid chromatography. J.Chromatogr., 1991, 565, 401-407 [plasma; SPE; pharmacokinetics; LOQ 31 nM] Lansdorp, D.; Janssen, T.J.; Guelen, PJ.; Vree, T.B. High-performance liquid chromatographic method for the determination of diclofenac and its hydroxy metabolites in human plasma and urine. J.Chromatogr., 1990, 528, 487-494 [plasma; urine; column temp 30; extracted metabolites; pharmacokinetics; LOD 20 ng/mL (plasma); LOD 2.5 |xg/mL (urine)] Lee, H.S.; Kim, E.J.; Zee, O.P.; Lee, YJ. High performance liquid chromatographic determination of diclofenac sodium in plasma using column-switching technique for sample clean-up. Arch.Pharm. (Weinheim)., 1989, 322, 801-806 Zecca, L.; Ferrario, P. Determination of diclofenac in plasma and synovial fluid by high-performance liquid chromatography with electrochemical detection. J.Chromatogr, 1989, 495, 303-308 [plasma; electrochemical detection] El-Sayed, Y.M.; Abdel-Hameed, M.E.; Suleiman, M.S.; Najib, N.M. A rapid and sensitive high-performance liquid chromatographic method for the determination of diclofenac sodium in serum and its use in pharmacokinetic studies. J.Pharm.Pharmacol, 1988, 40, 727-729 Godbillon, J.; Gauron, S.; Metayer, J.P. High-performance liquid chromatographic determination of diclofenac and its monohydroxylated metabolites in biological fluids. J.Chromatogr, 1985, 338, 151 159 Plavsic, F.; Culig, J. Determination of serum diclofenac by high-performance liquid chromatography by electromechanical detection. Hum.Toxicol., 1985, 4, 317-322 Said, S.A.; Sharaf, A.A. Pharmacokinetics of diclofenac sodium using a developed HPLC method. Arzneimittelforschung, 1981, 31, 2089-2092 Tammara, V.K.; Narurkar, M.M.; Crider, A.M.; Khan, M.A. Morpholinoalkyl ester prodrugs of diclofenac: Synthesis, in vitro and in vivo evaluation. J.Pharm.Sci., 1994, 83, 644-648 [rat; plasma; mefenamic acid (IS)]