HPLCVARIABLES

Column: 250 X 4.6 Zorbax SAX Mobile phase: MeOH: buffer 50:50 (Buffer was 180 mM Na2HPO4 adjusted to pH 3.00 0.05 with 180 mM orthophosphoric acid. Pass mobile phase through a 250 X 4.6 25-40 [Lin silica (HPLC Technology) column to saturate it with silica.) Flow rate: 1 Detector: UV 253
CHROMATOGRAM Retention time: 9.5 OTHER SUBSTANCES

Simultaneous: acetaminophen, albuterol, aspartame, aspirin, beclomethasone dipropionate, caffeine, isoproterenol, menthol, minocromil, nedocromil, quinoline yellow, reproterol, riboflavin, saccharin, salicylic acid, sorbitan trioleate, terbutaline, theophylline
REFERENCE
Baker, RR.; Gardner, J.J.; Wilkinson, D. Automated high-performance liquid chromatographic method for the determination of nedocromil sodium in human urine using bimodal column switching. J.Chromatogr.B, 1995, 668, 59-65

SAMPLE

Matrix: urine Sample preparation: 10 mL Urine + 5 g NaCl + 1 mL water + 1 mL concentrated HCl + 10 mL diethyl ether, shake for 10 min at 200 oscillations/min, centrifuge at 1540 g for 10 min. Repeat extraction. Combine extracts, add 1 mL 1 M pH 3.5 glycine HCl buffer, shake, centrifuge, inject aliquot of lower aqueous phase
HPLCVARIABLES

Column: 250 X 4.6 10 |xm Partisil SAX Mobile phase: 0.9 M Orthophosphoric acid adjusted to pH 2.30 0.01 with 5 M NaOH Flow rate: 3.6 Injection volume: 120 Detector: UV 325
CHROMATOGRAM

Retention time: 4.5 Limit of detection: 50 ng/mL

OTHER SUBSTANCES

Noninterfering: acetaminophen, aspirin, hydrocortisone, phenylbutazone, prednisolone, salicylic acid, terbutaline, theophylline
REFERENCE
Gardner, J.J. Determination of sodium cromoglycate in human urine by high-performance liquid chromatography on an anion-exchange column. J.Chromatogr., 1984, 305, 228-232

Cyclobenzaprine
Molecular formula: C20H21N Molecular weight: 275.4 CAS Registry No.: 303-53-7 (cyclobenzaprine), 6202-23-9 (cyclobenzaprine hydrochloride)

SAMPLE Matrix: blood Sample preparation: 1 mL Serum + 500 |JIL 250 |xg/mL protriptyline hydrochloride + 1 mL 500 mM NaOH + 4 mL toluene: n-hexane: isoamyl alcohol 77:22:3, mix for 10 min, centrifuge at 3000 rpm for 5 min. Remove the upper organic layer and evaporate it to dryness under a stream of air at 40, reconstitute the residue in 200 |xL MeOH, inject a 50 |xL aliquot. HPLCVARIABLES Column: 150 X 4.7 5 fim Supelcosil LC-PCN cyanopropyl Mobile phase: MeCN: MeOH: 10 mM pH 7.2 potassium phosphate buffer 60:15:25 (Prepare buffer by mixing 194 mL 1.36 g/L KH2PO4 with 274 mL 1.74 g/L K2HPO4.) Flow rate: 2 Injection volume: 50 Detector: UV 254 CHROMATOGRAM Retention time: 3.7 Internal standard: protriptyline (8.1) OTHER SUBSTANCES Extracted: norcycyclobenzaprine, nortriptyline Interfering: amitriptyline KEYWORDS serum REFERENCE
Wong, E.C.C; Koenig, J.; Turk, J. Potential interference of cyclobenzaprine and norcyclobenzaprine with HPLC measurement of amitriptyline and nortriptyline: resolution by GC-MS analysis. JAnal.ToxicoL, 1995, 19, 218-224

SAMPLE Matrix: blood Sample preparation: 2 mL Plasma + 500 JJLL 10 \xg/mL nortriptyline hydrochloride in water + 500 (xL 100 mM HCl, mix, add 500 |xL saturated sodium bicarbonate, vortex briefly, when effervescence ceases add 10 mL dichloromethane: pentane 30:70, shake for 25 min, centrifuge at 4 at 1400 g for 25 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at room temperature, reconstitute the residue in 50 fxL mobile phase, inject the whole amount. HPLCVARIABLES Guard column: Resolve Si Guard-PAK (Waters) Column: 300 X 3.9 10 |xm ixPorasil Mobile phase: MeCN: EtOH:tert-butylamine 10:90:0.025 Flow rate: 1

Injection volume: 50 Detector: UV 230

CHROMATOGRAM

Retention time: 7.5 Internal standard: nortriptyline (13.7) Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES

Noninterfering: acetaminophen, aspirin, caffeine, ibuprofen

KEYWORDS

plasma; normal phase; use silanized glassware

REFERENCE
Hwang, P.T.R.; Young, D.A.; Straughn, A.B.; Meyer, M.C. Quantitative determination of cyclobehzaprine in human plasma by high pressure liquid chromatography. J.Liq.Chromatogr., 1993, 16, 1163-1171

SAMPLE

Matrix: blood, urine Sample preparation: Plasma. 1 mL Plasma + 100 |xL 500 ng/mL trimipramine in MeOH + 1 mL 200 mM pH 9.8 carbonate buffer + 5 mL hexane, rotate for 15 min, centrifuge at 3000 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 300 |xL mobile phase, inject a 150 jxL aliquot. Urine. 1 mL Urine + 1 mL p-glucuronidase (7200 Fishman Units) in 20 mM pH 6.5 phosphate buffer, heat at 37 for 24 h, add 100 p,L 10 M NaOH, add 5 mL hexane, rotate for 15 min, centrifuge at 3000 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 300 J U L L mobile phase, inject a 150 |JLL aliquot.
HPLCVARIABLES

Guard column: 20 mm long C18 base-deactivated silica (BDS) (Keystone) Column: 250 X 4.6 5 |xm C18 base-deactivated silica (BDS) (Keystone) Mobile phase: MeCN.buffer 50:50 (plasma) or 43:57 (urine) (Buffer was 0.085% phosphoric acid adjusted to pH 6.5 with triethylamine.) Flow rate: 1 Injection volume: 150 Detector: UV 229
CHROMATOGRAM

Retention time: 7.8 (plasma), 9.6 (urine) Internal standard: trimipramine (10.5 (plasma), 12.8 (urine)) Limit of quantitation: 0.5 ng/mL (plasma); 10 ng/mL (urine)
KEYWORDS

plasma
REFERENCE
Constanzer, M.; Chavez, C; Matuszewski, B. Development and comparison of high-performance liquid chromatographic methods with tandem mass spectrometric and ultraviolet absorbance detection for the determination of cyclobenzaprine in human plasma and urine. J.Chromatogr.B, 1995, 666, 117-126

SAMPLE

Matrix: blood, urine

Sample preparation: Plasma. 1 mL Plasma + 100 JJLL 500 ng/mL trimipramine in MeOH + 1 mL 200 mM pH 9.8 carbonate buffer + 5 mL hexane, rotate for 15 min, centrifuge at 3000 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 150 |xL mobile phase, inject a 75 |xL aliquot. Urine. 1 mL Urine + 1 mL (3-glucuronidase (7200 Fishman Units) in 20 mM pH 6.5 phosphate buffer, heat at 37 for 24 h, add 100 |xL 10 M NaOH, add 5 mL hexane, rotate for 15 min, centrifuge at 3000 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 1 mL mobile phase, inject a 50 \xL aliquot. HPLCVARIABLES Guard column: 20 X 4.6 C18 base-deactivated silica (BDS) (Keystone) Column: 50 X 4.6 5 |xm C18 base-deactivated silica (BDS) (Keystone) Mobile phase: MeCN: water 90:10 containing 0.1% formic acid and 10 mM ammonium acetate Flow rate: 1 Injection volume: 50-75 Detector: MS, PE Sciex API III, heated nebulized interface, corona discharge needle +4 IJLA, nebulizer probe 500, nebulizing gas was air at 2 L/min and 80 psi, curtain gas flow was nitrogen at 0.9 L/min, sampling orifice +45 V, dwell time 400 ms, interface heater 60, electron multiplier-3.7 kV, collision gas was argon 355 X 1012 atoms/cm2, first quadrupole filter admits m/z 276 (cyclobenzaprine) and 295 (trimipramine, collisional fragmentation at second filter, monitor m/z 215 (cyclobenzaprine) and 208 (trimipramine) at third quadrupole filter CHROMATOGRAM Retention time: 1.9 Internal standard: trimipramine (2.2) Limit of quantitation: 0.1 ng/mL (plasma); 10 ng/mL (urine) KEYWORDS plasma; pharmacokinetics REFERENCE
Constanzer, M.; Chavez, C; Matuszewski, B. Development and comparison of high-performance liquid chromatographic methods with tandem mass spectrometric and ultraviolet absorbance detection for the determination of cyclobenzaprine in human plasma and urine. J.Chromatogr.B, 1995, 666, 117-126

SAMPLE Matrix: formulations Sample preparation: Powder tablets (60 mesh), shake with 25 mL 25 mM sulfuric acid for 1 h, add MeOH to 85 mL, swirl, allow to cool to room temperature, make up to 100 mL. Remove a 10 mL aliquot, add 5 mL 1 mg/mL naphazoline hydrochloride in MeOH, make up to 100 mL, filter (0.45 |xm), inject 20 |xL aliquot. HPLC VARIABLES Column: 250 X 4.6 Zorbax C8 Mobile phase: MeCN: buffer 75:25 (Buffer was 12 g KH2PO4 in 1800 mL water, adjust pH to 3.0 with 1:3 phosphoric acid, make up to 2 L.) Flow rate: 1.5 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 6.8 Internal standard: naphazoline hydrochloride

OTHER SUBSTANCES Simultaneous: desipramine Interfering: amitriptyline KEYWORDS tablets REFERENCE

Heinitz, M.L. Determination of cyclobenzaprine in tablets by high-performance liquid chromatography. J.Pharm.ScL, 1982, 71, 656-658

SAMPLE Matrix: solutions HPLCVARIABLES Column: 150 X 2 Deltabond C8 (Keystone) Mobile phase: MeCN: 2-butanone: 50 mM pH 7.0 phosphate buffer 27:13:60 Flow rate: 0.15 Injection volume: 1 Detector: Chemiluminescence following post-column reaction. A 1 mM solution of Ru(2,2'bipyridine)32+ in 50 mM sodium sulfate (continuously sparged with helium) was oxidized to Ru(2,2'-bipyridine)33+ using a Princeton Applied Research Model 174A polarographic analyzer with a platinum gauze working electrode, a platinum wire auxiliary electrode, and a silver wire reference electrode. The Ru solution was pumped at 0.3 mL/min and mixed with the column effluent in the flow cell of the detector, a fluorescence detector with the light source removed. CHROMATOGRAM Retention time: 6 Limit of detection: 0.1-1 |xg/mL OTHER SUBSTANCES Simultaneous: dicyclomine KEYWORDS post-column reaction REFERENCE
Holeman, J.A.; Danielson, N.D. Microbore liquid chromatography of tertiary amine anticholinergic pharmaceuticals with tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection. J.Chromatogr. ScL, 1995, 33, 297-302

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 5 juim Supelcosil LC-DP (A) or 250 X 4 5 \xm 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: 15.50 (A), 7.05 (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, 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, 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, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, 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: solutions Sample preparation: Inject an aliquot of a solution in mobile phase. HPLC VARIABLES Column: 250 X 4.6 Econosil C8 Mobile phase: MeCN: buffer 30:70 (Buffer was 20 mM KH2PO4 and 14 mM triethylamine adjusted to pH 3.0 with phosphoric acid.) Injection volume: 20 Detector: UV 210

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CHROMATOGRAM

Retention time: 8.6 Limit of quantitation: <1 jxg/mL

OTHER SUBSTANCES

Simultaneous: doxepin, desipramine, protriptyline, maprotiline Also analyzed: amitriptyline, amoxapine, carbamazepine, imipramine, nortriptyline
KEYWORDS

UV spectra given
REFERENCE
Ryan, T.W. Identification and quantification of tricyclic antidepressants by UV-photodiode array detection with multicomponent analysis. J.Liq.Chromatogr., 1993, 16, 1545-1560

ANNOTATED BIBLIOGRAPHY

Puopolo, P.R.; Flood, J.G. Detection of interference by cyclobenzaprine in liquid- chromatographic assays of tricyclic antidepressants. Clin.Chem., 1987, 33, 819-820 [LOD 10 ng/mL] Schneider, M.; Giardina, E.-G.V. Inteference by Flexeril, a tricyclic muscle relaxant, with liquid-chromatographic determination of imipramine. Clin.Chem., 1986, 32, 1599 [plasma; interfering imipramine]