HPLC Analysis of Amitriptyline

7/28/2019 HPLC Analysis of Amitriptyline

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SAMPLEMatrix: bloodSample preparat ion: 1 mL S erum + 500 jxL 750 mM pH 10 sodium bicarbon ate/carbon atebuffer + 50 |xL IS in EtO H : w ater 50 :50 + 8 mL he pt an e: isoamyl alcohol 98 :2 , sh ake at250 cycles/min for 5 min, centrifuge at 1500 g for 10 m in, freeze in dry ice/EtO H. Rem ovethe organic layer and add it to 150 |xL 22 mM pH 2.5 KHaPCVphosphoric acid buffer,sh ake at 250 cycles/min for 5 min, centrifuge at 1500 g for 10 m in, freeze in dry ice/EtO H.Discard the organic layer, inject a 65 |xL aliquot of the aqueous layer.

HPLCVARIABLESColumn: 250 X 4.6 Supelco C18Mobile phase: M eCN : buffer 45 :55 (Buffer was 44 mM K H2PO 4 containing 1.5 mL/L trie-thylamine, adjusted to pH 2.5 with phosphoric acid.)Flow ra te : 1.5Inject ion volum e: 65Detector: UV 240CHROMATOGRAMRetent ion t ime: 11.5Internal s tandard: 1 -(3 -(dimethylamino)propyl) -1 -(p-chlorophenyl) -1,3-dihydroisobenzo-furan-5-carbonitrile (LU 10-202) (Lundbeck, Copenhagen) (8.33)OTHER SUBSTANCESExtracted: citalopram, nortriptylineSimul taneous: chlorprothixene, clomipramine, clozapine, flupenthixol, haloperidol, levo-mepromazine, perphenazine, zuclopenthixolNoninterfer ing : benzodiazepinesInterfering: didesmethylclomipramine, levomepromazineKEYWORDSserumREFERENCEOlesen, O.V.; Linnet, K. Simplified high-performance liquid chromatographic method for the determi-nation of citalopram and desmethylcitalopram in serum without interference from commonly usedpsychotropic drugs and their metabolites. J.Chromatogr.B, 1996, 675, 8 3 - 8 8SAMPLEMatrix: bloodSample preparat ion: 1 mL Pl asm a -I- 500 |xL 1 M sodium carb on ate, vo rtex for 3 0 s, add5 mL diethyl ether, vortex for 2 min, centrifuge at 4000 rpm for 10 min. Remove theorganic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitutethe residue in 250 fxL mobile phase, vortex for 30 s, centrifuge at 12000 rpm for 5 min,inject an aliquot of the supernatant.HPLCVARIABLESColumn: 300 X 3.9 10 |xm jxBondapak C18

AmitriptylineMolecular form ula: C20H23NMolecular weight: 277.4CAS R egistry No.: 50-48-6 (amitriptyline), 549-18-8 (amitriptylinehydrochloride)

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M obi le phase: M eOH : wa ter 6 2:3 8 adjusted to pH 3.5 with phosphoric acidColumn temp erature: 40F lo w rate : 1.2D etec tor: UV 262CHROMATOGRAMRetent ion t ime: 6 .1Internal s tandard: amitriptylineOTHER SUBSTANCESExtracted: pheniramineSimul taneous: chlorpheniramine, diazepam, diltiazem, flurbiprofen, ibuprofen, itracona-zole, ketoprofen, mebeverine, metoclopramide, phenylbutazoneKEYWORDSdog; plasma; amitriptyline is ISREFERENCEEl-Sayed, YM.; Niazy, E.M.; Khidir, S.H. High-performance liquid chromatographic method for thequantitative determination of pheniramine in plasma. J.Liq.Chromatogr., 1995, 18, 763777SAMPLEMatrix: bloodSample preparat ion: 1 mL Plas m a + doxepin + NaO H -f h ex an e: isoamyl alcohol 98 :2,extract. Remove the organic ph ase and add it to 0.03% phosphoric acid, extract, inject an

aliquot of the aqueous phase.HPLCVARIABLESGuard co lumn: C18Column: 100 X 8 10 jxm Resolve C8 (Waters)Mobile phase: MeC N: M eOH: 56 mM amm onium acetate :1 M amm onium hydroxide 100:10:4.5:2.6Flow rate: 2.5Detector: UV 220CHROMATOGRAMRetent ion t ime: 17.8Internal s tandard: doxepin (11.6)OTHER SUBSTANCESExtracted: fluoxetine, norfluoxetine, nortriptylineKEYWORDSplasmaREFERENCEel-Yazigi, A.; Chaleby, K.; Gad, A.; Raines, D.A. Steady-state kinetics of fluoxetine and amitriptyline inpatients tre ated with a combination of these drug s as compared with those treated with am itriptylinealone. J.Clin.PharmacoL, 1995, 35, 1 7 - 2 1SAMPLEMatrix: bloodSample preparat ion: 2 mL Whole blood or pla sm a + 2 mL buffer + 5 mL chloroform:isopropanol: n-h eptan e 6 0: 14 :2 6, sh ake gen tly horizontally for 10 min, centrifuge at 2800g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum


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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 chloridesolution 25% diluted w ith water, adjusted to pH 9.5 with 25% amm onia solution.)HPLCVARIABLESColumn: 300 X 3.9 4 |xm NovaPack C18Mobi le phase: M eOH :TH F:buffer 65 :5 :3 0 (Buffer w as 0.68 g/L (10 mM (sic)) KH 2PO 4adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each sessionwa sh th e column with w ater for 1 h an d MeOH for 1 h, re-equilibrate for 30 m in.)Column temperature: 30Flow rate: 0 .8Inject ion volum e: 50Detector: UV 240CHROMATOGRAMRetent ion t ime: 9.23Limit of detect ion:


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REFERENCETracqui, A.; Kintz, P.; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic ScL,1995, 40, 254-262SAMPLEMatrix: bloodSample preparat ion: 1 mL Serum + 500 |JLL 250 |xg/mL protriptyline hydrochloride + 1mL 500 mM NaOH + 4 mL tolue ne: n-hexa ne: isoamyl alcohol 7 7:2 2: 3 , mix for 10 min,centrifuge at 3000 rpm for 5 min. Remove the upper organic layer and evaporate it todryness under a stream of air at 40, reconstitute the residue in 200 |JLL MeOH, inject a50 |xL aliquot.HPLCVARIABLESColumn: 150 X 4.7 5 jxm Supelcosil LC-PCN cyanopropylMobile phase: MeCN.MeOH.10 mM pH 7.2 potassium phosphate buffer 60:15:25'(Pre-

pare buffer by mixing 194 mL 1.36 g/L KH 2PO 4 with 274 mL 1.74 g/L K 2HPO4.)Flow rate: 2Inject ion volume: 50Detector: UV 254CHROMATOGRAMRetention time: 3.8Internal s tandard: protriptyline (8.1)OTHER SUBSTANCESExtracted: norcycyclobenzaprine, nortriptylineInterfering: cyclobenzaprineKEYWORDSserumREFERENCEWong, E.C .C ; Koenig, J .; Turk, J . P otentia l interference of cyclobenzaprine an d norcyclobenzaprine withHPLC measurement of amitriptyline and nortriptyline: resolution by GC-MS analysis.J.Anal.ToxicoL, 1995, 19, 218-224SAMPLEMatrix: bloodSample preparat ion: Automated SPE by ASPEC system. Condition a C18 Clean-Up SPEcartridge (CEC 18111, Worldwide Monitoring) with 2 mL MeOH then 2 mL water. 1 mLPlasma + 1 mL 400 ng/mL protriptyline in water, vortex, add to SPE cartridge, washwith 3 mL water, wash with 3 mL 750 mL/L methanol. Elute with three aliquots of 300IxL 0.1 M amm onium acetate in MeOH . Add 0.5 mL 0.5 M NaO H and 4 mL 50 mL/Lisopropanol in heptane to eluate, mix thoroughly. Allow 5 min for phase separation. Re-

move upper heptane phase and add it to 300 jxL 0.1 M phosphoric acid (pH 2.5), mix,separate, inject a 100 |xL aliquot of the aqueous phase.HPLCVARIABLESGuard co lumn: LC-8-DB (Supelco)Column: 150 X 4.6 LC-8-DB (Supelco)Mobile phase: M eCN : buffer 3 5:6 5 (Buffer w as 10 mL/L triethylam ine in w ater adjustedto pH 5.5 with glacial acetic acid.)Flow rate: 2Inject ion volume: 100Detector: UV 228


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CHROMATOGRAMRetent ion t ime: 5 .6Internal s tandard: protriptyline (4)OTHER SUBSTANCESExtracted: chlordiazepoxide, chlorimipramine, chlorpromazine, desipramine, dextrome-thorphan, diazepam, diphenhydramine, doxepin, encainide, fentanyl, flecainide, fluoxe-t ine, flurazepam, haloperidol, hydroxyethylfiurazepam, ibuprofen, im ipram ine, lidocaine,maprotiline, methaqualone, mexiletine, midazolam, norchlorimipramine, nordoxepin, nor-diazepam, nortriptyline, norverapamil, pentazocine, promazine, propafenone, propoxy-phene, propranolol, protriptyline, quinidine, trazodone, verapamilNoninterfer ing : acetaminophen, acetylmorphine, amiodarone, amo barbital, amph etam ine,bendroflumethiazide, benzocaine, benzoylecgonine, benzthiazide, butalbital, carbamaze-pine, chlorothiazide, clonazepam, cocaine, codeine, cotinine, cyclosporine, cyclothiazide,desalkylflurazepam, diamorphine, dicumerol, ephedrine, ethacrynic acid, ethanol, eth-chlorvynol, ethosuximide, furosemide, glutethimide, hydrochlorothiazide, hydrocodone,

hydroflumethiazide, hydromorphone, lorazepam, mephentermine, meprobamate, meth-amphetamine, metharbital, methoxsalen, methoxyphenteramine, methsuximide, meth-ylcyclothiazide, metoprolol, MHPG, monoacetylmorphine, morphine, normethsuximide,oxazepam, oxycodone, oxymorphone, pentobarbital, phencyclidine, phenteramine, phen-ylephrine, phenytoin, polythiazide, primidone, prochlorperazine, salicylic acid, sulfanila-mide, THC-COOH, theophylline, thiazolam, thiopental, thioridazine, tocainide, trichlo-romethiazide, trifluoperazine, valproic acid, warfarinInterfering: acetazolamide, methadone, norfluoxetine, tem azepam , trimip ram ineKEYWORDSSPE; plasmaREFERENCENichols, J.H.; Charlson, J.R.; Lawson, G.M. Automated HPLC assay of fluoxetine and norfluoxetine inserum. Clin.Chem., 1994, 40, 1312-1316SAMPLEMatrix: bloodSample preparat ion: Condition a 1 mL Bond Elut C18 SPE cartridge with 1 mL 1 M HCl,1 mL MeOH, 1 mL water, and 1 mL 1% potassium carbonate. 700 JJLL Serum + 50 |xL 5|xg/mL trimip ram ine in 5% potas sium bicarbo nate + 700 |xL MeCN , vortex, centrifuge at1500 g for 5 min, add supernatant to SPE cartridge (at ca. 1 mL/min). Wash with 2 mLwater and 1 mL MeCN, elute with 250 |JLL M eOH : 35% perchloric acid 2 0: 1 by gravity(10 min) then centrifuge for 20 s to remove rest of eluant, inject a 50 jxL aliquot of theeluHPLCVARIABLESGuard co lumn : 15 mm 7 jxm Brownlee RP-8Column: 150 X 4.6 5 \xm Ultrasphere OctylMobile phase: M eCN: wa ter 37.5:62.5 con taining 0.5 g/L tetrame thylam m onium perchlo-rate and 0.5 mL/L 7% perchloric acidF low rate: 1.5Inject ion volum e: 50Detector: UV 215CHROMATOGRAMRetent ion t ime: 8 .3Internal s tandard: trimipramine (9.6)Limit of quant i tat ion: 5 ng/mL


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OTHER SUBSTANCESExtracted: clomipramine, desipramine, doxepin, fluoxetine, fluvoxamine, imipramine, nor-triptyline, protriptylineInterfering: desmethyltrimipramine, maprotilineKEYWORDSSPE; serumREFERENCEGupta, R.N. An improved solid phase extraction procedure for the determination of antidepressants inserum by column liquid chromatography. J.Liq.Chromatogr., 1993, 16, 2751-2765SAMPLEMatrix: bloodSample preparat ion: 500 |xL Serum + 250 |xL 100 mM lauryl sulfate, centrifuge at 2500

g for 8 min, inject a 250 |JLL aliquot of the sup ern atan t onto column A with m obile phaseA, elute with mobile phase A for 6 min, backflush contents of column A onto column Bwith m obile pha se B for 4 min. E lute column B with mobile ph ase B for 6 min a nd conductanalysis. When not in use flush column A with mobile phase A. Every eight injectionsbackflush column A with M eCN: wa ter 70:3 0.HPLCVARIABLESColumn: A Guard-Pak 10 |xm Resolve CN (Waters); B 150 X 3 7 jim Separon SGX CN(Tessek)Mobile phase: A M eCN: w ater 3 :97; B M eCN : buffer 26:7 4 (Buffer wa s 50 mM phosphoric

acid, 50 mM ammonium phosphate, and 28 mM diethylamine, pH 2.55.)Flow rate: 1Inject ion volume: 250Detector: UV 210CHROMATOGRAMRetent ion t ime: 12Limit of detect ion: 20-25 ng/mLOTHER SUBST ANC ESExtracted: nortriptylineKEYWORDScolumn-switching; serumREFERENCEDolezalova, M. On-line solid-phase extraction and high-performance liquid chromatographic determi-nation of nortriptyline and amitriptyline in serum. J.Chromatogr., 1992, 579, 291-297SAMPLEMatrix: bloodSample preparat ion: For each 1 mL p lasm a or serum add 10 jxL 14 |xg/mL t rim ipra m inein MeOH. Inject serum or plas m a directly onto column A with mobile ph ase A, elute w ithmobile phase A to waste. After 15 min elute column A onto column B (foreflush) withmobile phas e B. After 2 min rem ove column A from th e circuit, elut e column B w ith m obilephas e B, monitor the effluent from column B. Re-equilibrate column A with mobile ph aseA.HPLCVARIABLESColumn: A 20 X 4.6 10 |xm Hypersil MOS C8; B 20 X 4.6 5 jxm Hypersil CPS CN + 250

X 4.6 5 \xm Nucleosil 100 CN


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Mobile phase: A M eOH: water 5:95; B MeC N: M eOH : buffer 578 :188:23 5 (Buffer was 10mM K 2HPO 4 adjusted to pH 6.8 w ith 85% phosphoric acid.)F lo w rate: 1.5Inje ction volu m e: 100D etecto r: UV 214CHROMATOGRAMRetent ion t ime: 8.54Internal s tandard: t r imipramine (6.5)Limit of detect ion: 5-10 ng/mL (1 ng/mL with 3 injections)OTHER SUBSTANCESExtracted: metabolites, desipramine, fluvoxamine, imipramine, maprotiline, nortriptylineNoninterfer ing : chlordiazepoxide, clobazam, clozapine, diazepam, flurazepam, fluspiri-lene, haloperidol, nitrazepam, oxazepam, perazine, pimozide, spiroperidol, trifluperidolInterfering: clomipramine, doxepinKEYWORDSplasma; serum; column-switchingREFERENCEHartter, S.; Hiemke, C. Column switching and high-performance liquid chromatography in the analysisof amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum.J.Chromatogr., 1992, 578, 273-282

SAMPLEMatrix: bloodSample preparat ion: 1 mL Serum + 3 (xL 20 ng/mL clobazam in methanol -I- 1 mL sat-urated sodium borate (pH adjusted to 11 with 6 M NaOH) + 5 mL n-hexane, mix for 2min, centrifuge at 3 000 g for 10 min. Se par ate organic phase , evaporate to dryness u nd era s trea m of helium at 30, rec on stitu te in 20 jxL mob ile ph ase , inject a 10 |xL aliquot.HPLCVARIABLESGuard co lumn: 20 mm 40 jxm Pelliguard LC-8 (Supelco)Column: 150 X 4.6 Supelco 5 |xm C8Mobile phase: M eCN : buffer 50 :50 (Buffer w as 10 mM N aH 2PO 4 + 1.2 mL/L butylamine,pH adjusted to 3 with phosphoric acid.)Flow rate: 1Inject ion volum e: 10Detector: UV 254CHROMATOGRAMRetent ion t ime: 9Internal s tandard: clobazam (5)Limit of detect ion: 10 ng/mLOTHER SUBSTANCESExtracted: clomipramine, desipramine, imipramine, nortriptylineSimul taneous: alprazolam, clonazepam, diazepam, flunitrazepam, haloperidol, lorazepam,maprotiline, nitrazepam, triazolamKEYWORDSserum


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REFERENCESegatti, M.P.; Nisi, G.; Grossi, R; Mangiarotti, M.; Lucarelli, C. Rapid and simple high-performanceliquid chromatographic determination of tricyclic antidepressants for routine and emergency serumanalysis. J.Chromatogr., 1991, 536, 3 1 9 - 3 2 5

SAMPLEMatrix: bloodSample preparat ion: 1 mL Plasma + 20 |xL 10 |Jig/mL clovoxamine + 120 |xL 2 M NaOH+ 4 mL hep tan e: isopropanol 98 :2, sh ake for 30 min, centrifuge at 3 000 g for 10 min.Remove the organic layer and add it to 100 |xL 100 mM HCl, shake for 20 min, centrifugeat 3000 g for 10 min, inject a 20 |xL aliquot of the aqueous layer.HPLCVARIABLESColumn: 120 X 4.6 5 |xm Nucleosil C8Mobi le phase: M eCN:buffer 3 6:64 (Buffer w as 16 mM KH 2PO 4 adjusted to pH 2.5 withconcentrated phosphoric acid.)Flow rate: 1Inject ion vo lum e: 20Detector: UV 215CHROMATOGRAMRetent ion t ime: 5 .5Internal s tandard: clovoxamine (3.3)

OTHER SUBSTANCESExtracted: chlorimipramine, desipramine, doxepin, fluvoxamine, imipramine, nortripty-line, t r imipramineKEYWORDSplasmaREFERENCEFoglia, J.P.; Birder, L.A.; Perel, J.M. Determination of fluvoxamine in human plasma by high-perfor-mance liquid chromatography with ultraviolet detection. J.Chromatogr., 1989, 495, 2 9 5 - 3 0 2SAMPLEMatrix: bloodSample preparat ion: Inject 200 |xL serum onto column A and elute with mobile phase Afor 10 min the n back-flush column A onto column B w ith mobile phase B for 4 m in. E lutecolumn B with mobile ph ase B and m onitor th e effluent. Remove column A from circuitand w ash w ith MeC N: w ater 6 0:40 for 6 min th en w ith mobile ph ase A for 10 min.HPLCVARIABLESColumn: A 40 X 4 TSKprecolumn PW (Tosoh); B 150 X 4 TSKgel ODS-80TM (Tosoh)Mobile phase: A 50 mM pH 7.5 potassium phosph ate; B MeCN : 100 mM pH 2.7 potassiumpho spha te 32.5 :67.5, containing 0.2 g/L sodium 1-heptanesulfonateFlow rate: 1Inject ion volume: 200Detector: UV 210CHROMATOGRAMRetent ion t ime: 17

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HPLC Analysis of Amitriptyline