Nor Trip Ty Line

8/17/2019 Nor Trip Ty Line

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SAMPLE

Matrix: blood

Sample p repa ra ti o n : 1 mL Serum + 3 mL MTBE, vortex for 1 min, centrifuge at 1500

rpm for 5 min. Remove the organic layer and evaporate it to dryness under a stream of

nitrogen at room temperature, reconstitute the residue in 100 jxL MeOH, inject an aliquot.

HPLCVARIABLES

G uar d co lum n: 18 mm long (Brownlee)

Column: 300 X 3.6 10 |xm jxBondapak C18

Mobile ph as e: MeOH: water 80:20

Flow rate: 1

Dete cto r: UV 254; RIA

C H R O M A T O G R A M

Retention time: 5.6

OTHER SUBSTANCES

Extracted: metabolites, norgestimate

KEYWORDS

serum

REFERENCE

Wong, F.A.; Juzw in, S.J.; Tischio, N.S.; Flor, S.C. Determ ination of norgestim ate in serum by a utom ated

high-performance liquid chromatography and subsequent radioimmunoassay. J.Liq.Chromatogr.

1995,28, 1851-1861

SAMPLE

Matrix: bulk

HPLCVARIABLES

Colu mn: 250 X 4 10 |xm LiChrosorb RP-18

Mobile ph as e: MeOH: water 70:30

Flow rate: 1

Injection volume: 25

D etecto r: UV 240


Retention t ime: 9

OTHER SUBSTANCES

Simultaneous: impurities, norethindrone

Interfering: ethinyl estradiol

REFERENCE

Gorog, S.; Hereny i, B. An alysis of stero ids. XXXVIII. The u se of high-perform ance liquid chro m atog raph y

with diode-array UV detection for es tim ating imp urity profiles of steroid drug s. J.Chromatogr. 1987,

400 177-186

Norgestrel

Molecular formula: C

21

H

28

O

2

Molecular weight: 312.5

CAS R egistry No.: 797-63-7, 797-64-8 ((-) form), 6533-00-2

Previous page

http://81765_13b.pdf/http://81765_13b.pdf/


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SAMPLE

Matrix: culture medium

Sample preparat ion: Extract culture medium twice with 2 volumes of ether, combine the

extracts and evaporate them to dryness, reconstitute with MeOH, inject an aliquot.

HPLCVARIABLES

Column: 300 X 3.9 Techopak 10 C18 (HPLC Technology)

Mobile phase:

MeOH: water 70:30

F low rate: 1.5

Detector:

UV 240; Radioactivity

CHROMATOGRAM

Retent ion t ime: 5

OTHER SUBSTANCES

Extracted:

metabolites, norgestimate

KEYWORDS

tritium labeled

REFERENCE

Wild, M.J.; Rudland, RS.; Back, D.J. Metabolism of the oral contraceptive steroids ethynylestradiol and

norge stima te by norm al (Hum a 7) and m align ant (MCF-7 and ZR-75-1) hu m an b rea st cells in culture.

J.Steroid Biochem.Mol.BioL 1991, 39 5 3 5 - 5 4 3

SAMPLE

Matrix:

formulations

Sample preparat ion: Centrifuge oil formulation at 30° at 2000 rpm for 30 min, filter

(W hatman No. 1 paper), collect the last 4 mL of th e n itra te. D ilute a 10 |xL aliquot to 10

mL with M eCN : w ater 60 :40 containin g 0.3% Tween 80, remove a 2 mL aliquot a nd add

it to 1 mL 3.33 |xg/mL progesterone, vortex for 10 s, inject a 50 |xL aliquot.

HPLCVARIABLES

Column:

300 X 3.9 Novapak C18

Mobile phase: MeCN: water 60:40

Flow rate: 2

Inject ion volume: 50

Detector: UV

248

CHROMATOGRAM

Internal s tandard:

progesterone

KEYWORDS

oils

REFERENCE

Gao, Z.-H.; Shu kla, A. J.; Joh nso n, J.R.; Crowley, W.R. Controlled re leas e of a con tracep tive ste roid from

biodegradable and injectable gel formulations: In vitro evaluation. Pharm .Res. 1995, 12 857—863

SAMPLE

Matrix:

formulations

Sample preparat ion: Oils. 1 mL Sam ple + 25 mL MeO H-.water 90: 10 , sha ke vigorously

for 5 min, centrifuge, inject a 10

|JLL

aliquot of the supernatant. Tablets. Grind a tablet

to a fine powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 |min), discard first

5 mL of filtrate, inject a 10 |xL aliquot of the remaining filtrate. Suspensions (aqueous).


3/36

Make up 5 mL to 50 mL with MeOH, filter (0.45 jjim), discard first 5 mL of filtra te, inject

a 10 |xL aliquot of the remaining filtrate.

HPLCVARIABLES

Column: 250 X 4.6 5 |xm Zorbax ODS

Mobile phase:

MeOH: water 75:25

Flow rate: 1.5


Detector: UV

240

CHROMATOGRAM

Retention time: 5.9

Limit of detection:

5 |xg/mL

OTHER SUBSTANCES

Simultaneous:

aspirin, benzyl alcohol, benzyl benzoate, boldenone, caffeine, caluster6ne,

cortisone, dehydroepiandrosterone (UV 210), fluoxym esterone, formebolone, mesterolone

(UV 210), methandriol (UV 210), methenolone acetate, methyltestosterone, mibolerone,

nandrolone acetate, nandrolone propionate, norethandrolone, norethindrone, norethin-

drone acetate, oxandrolone (UV 210), oxymetholone, stanozolol, testolactone, testosterone

acetate, testosterone propionate, trenbolone acetate

Interfering:

ethisterone, methandrostenolone, nandrolone, testosterone

KEYWORDS

oils;

tablets; suspensions

REFERENCE

Walters, M.J.; Ayers, R.J.; Brown, D.J. Analysis of illegally distributed anabolic steroid products by

liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotom-

etry J.Assoc.OffAnaLChem. 1990, 73 904-926

SAMPLE

Matrix:

microsomal incubations, mucosal fluid

Sample preparation:

Mucosal fluid . Extract 1 mL mucosal fluid twice with 5 mL diethyl

ether, evaporate extracts to dryness, resuspend residue in 100 |xL MeOH, inject an ali-

quot. Microsomal incubations. Extract 2.5 mL microsomal preparation with 5 mL diethyl

ether, proceed as before.

HPLCVARIABLES

Guard column:

on-line guard column

Column:

100 X 8 ixBondapak radial compression module

Mobile phase: MeOH: water 70:30

Flow rate: 1.5

Injection volume:

100

Detector: UV 240

CHROMATOGRAM

Retention time: 6

OTHER SUBSTANCES

Simultaneous:

3-ketonorgestimate, 17-deacetylnorgestimate, norgestimate

REFERENCE

Madden, S.; Back, D.J. Metabolism of norgestimate by human gastrointestinal mucosa and liver micro-

somes in vitro. J.Steroid Biochem.Mol.Biol. 1991, 38 4 9 7 - 5 0 3


4/36

SAMPLE

Matrix:

solutions

HPLC VARIABLES

Column:

50 X 4.6 5 |xm Supelcosil LC-18

Mobile phase:

MeOH: THF: water 10:20:70

Flow rate: 2


Detector: UV 220

CHROMATOGRAM

Retention time: 5.5

OTHER SUBSTANCES

Simultaneous: ethinyl estradiol, norethindrone, norethindrone acetate, norethynodrel

acetate

REFERENCE

Supelco Catalog Supelco

Inc.

Bellefonte

PA

1996, p. A130

SAMPLE

Matrix:

solutions

Sample preparation: Inject an aliquot of a 10 |xg/mL solution.

HPLC VARIABLES

Column: 150 X 4.6 Supelco ODS

Mobile phase:

MeCN: water 25:75 containing 14 mM cyclodextrin

Column temperature: 0

Flow rate: 1-3


Detector: UV 240

CHROMATOGRAM

Retention time:

16 D +)), 18 D -))

KEYWORDS

chiral

REFERENCE

Lamparczyk, H.; Zarzycki, RK.; Nowakowska, J. Effect of temperature on separation of norgestrel en-

antiomers

by

high-performance liquid chromatography. J.Chromatogr.A

1994,

668 413-417

SAMPLE

Matrix:

solutions

Sample preparation:

Direct injection.

HPLCVARIABLES

Column: 250 X 4.6 10 m Partisil C-18 ODS-3

Mobile phase:

MeCN-.water 50:50

Flow rate: 2

Detector: UV

243

CHROMATOGRAM

Retention time: 6.0


5/36

KEYWORDS

see also J.Pharm.Sci. 1989, 78, 477

REFERENCE

Catz, P.; Friend, D.R. In vitro evaluations of transdermal levonorgestrel. D rug D es.Deliv. 1990, 6 4 9 -

60

SAMPLE

Matrix: solutions

HPLCVARIABLES

Column: 250 X 4.6 10 jim Partisil ODS-3 C18

Mobile phase: MeCN-.water 50:50

Flow rate: 2

D etecto r: UV 243


Retention time: 6.0

REFERENCE

Friend, D.R.; C atz, P.; Heller, J.; O kagaki, M. Tran sderm al delivery of levonorgestrel IV : Ev aluation of

membranes.

J.Pharm.Sci.

1989,

78

477-480

SAMPLE

Matrix: solutions

HPLCVARIABLES

Guard column: Chromsep reverse phase guard column (Chrompack)

Column: 100 X 3 5 |xm Chromspher glass column

M obile ph as e: MeCN: water 35:65

Flow rate: 0.4

Detec tor: UV 247


Retention time: 20

OTHER SUBSTANCES

Sim ul tane ou s: nandrolone, trenbolone (at UV 340)

REFERENCE

Haa snoot, W.; Schilt, R.; Ha m ers , A.R.; Huf, F.A.; Farjam , A.; Fre i, R.W.; B rin km an , U.A. Dete rm ina tion

of p-19-nortestosterone a nd its metab olite a-19-nortestosterone in biological samples at th e sub pa rts

per billion level by high-performance liquid chromatography with on-line immunoaffinity sample

pretreatment. J.Chromatogr 1989, 489 157-171

SAMPLE

Matrix: solutions

HPLCVARIABLES

Colum n: 250 X 4.6 10 jxm Nucleosil C18

Mobile pha se : MeCN:THF: water 12.9:22.4:64.7

Flow rate: 1

D etecto r: UV 240


6/36


Retention t ime: 13

OTHER SUBSTANCES

Sim ul tane ous : estrone, ethinyl estradiol, mestranol, norethindrone, norethindrone acetate

REFERENCE

Gazdag, M.; Szepesi, G.; Szeleczki, E. Selection of high-performance liquid chromatographic methods in

pharmaceutical analysis. I. Optimization for selectivity in reversed-phase chromatography.

J.Chromatogr. 1988, 454 8 3 - 9 4

SAMPLE

Matrix: solutions

HPLCVARIABLES

Co lumn: 250 X 4.6 5

\xm

LiChrosorb Si 60

Mobile phase : Hexane: dioxane: isopropanol 95 : 3:2

Flow rate: 1

D etec tor: UV 254


Retention t ime: 13

OTHER SUBSTANCES

Sim ul tane ou s: estrone, ethinyl estradiol, mestranol, norethindrone, norethindrone acetate

KEYWORDS

normal phase

REFERENCE

Gazdag, M.; Szepesi, G.; Fabian-Varga, K. Selection of high-performance liquid chro ma tographic m eth-

ods in pharmaceutical analysis. II. Optimization for selectivity in normal-phase systems.

J.Chromatogr.

1988,

454 9 5 - 1 0 7

SAMPLE

Matrix: solutions

HPLCVARIABLES

Colum n: 250 X 4.6 jmBondapak C18

M obile phase: Dioxane: water 50:50 (Caution Dioxane is a carcinogen )

Flow rate: 1.4

D etec tor: UV 254

OTHER SUBSTANCES

Simultaneous: norgestimate

REFERENCE

Killinger, J.; Ha hn, D.W.; Phillips, A.; Hetyei, N.S.; McGuire, J.L. T he affinity of norgestim ate for u ter ine

progestogen receptors and its direct action on the uterus.

Contraception

1985,

32

311-319

SAMPLE

Matrix: tissue

Sa m ple p re pa ra ti o n : Incubate endometrial tissue with buffer, remove tissue, extract me-

dium twice with 2 volumes of diethyl ether, evaporate to dryness, reconstitute in a small

volume of MeOH, inject an aliquot.


7/36

HPLC VARIABLES

Column:

300

X 3.9

Technopak

10

C18

Mobile phase:

MeOH: water 70:30

Flow rate: 1.5

Detector: UV 240

CHROMATOGRAM

Retention time: 6

OTHER SUBSTANCES

Simultaneous: metabolites, norgestimate

KEYWORDS

endometrial tissue

REFERENCE

Wild,

M.J.;

Rudland,

RS.;

Back, D.J. M etabolism of the oral contraceptive steroids ethyny lestradiol,

norgestimate and 3-ketodeso gestrel by a hu m an endo me trial cancer cell line (HEC-IA) and endo-

metrial tissue in vitro. J.Steroid Bioche m.M ol.BioL 1993,

45

407-420


8/36

SAMPLE

Matrix:

blood

Sample preparat ion: 1 mL Se rum + 500 |xL 750 mM pH 10 sodium bicarbon ate/carbonate

buffer + 50 |JLL IS in EtO H: wa ter 50:50 + 8 mL hep tan e: isoamyl alcohol 98 :2, shake at

250 cycles/min for 5 min, centrifuge at 1500 g for 10 m in, freeze in d ry ice/EtOH . Remove

the organic layer and add it to 150 |xL 22 mM pH 2.5 KH

2

PO

4

/phosphoric acid buffer,

sh ak e a t 250 cycles/min for 5 m in, centrifuge at 1500 g for 10 m in, freeze in dry ice/EtOH .

Discard the organic layer, inject a 65 JJLL aliquot of the aqueou s layer.

HPLCVARIABLES

Column:

250 X 4.6 Supelco C18

Mobile phase: M eCN : buffer 45 :55 (Buffer wa s 44 mM KH

2

PO

4

containing 1.5 mL/L trie-

thylamine, adjusted to pH 2.5 with phosphoric acid.)

Flow ra te: 1.5

Inject ion volum e: 65

Detector:

UV 240

CHROMATOGRAM

Retention time: 9.73

Internal s tandard: l-(3-(dimethylamino)propyl)-l-(p-chlorophenyl)-l,3-dihydroisobenzo-

furan-5-carbonitrile (LU 10-202) (Lundbeck, Copenhagen) (8.33)

OTHER SUBSTANCES

Extracted:

metabolites, amitriptyline, citalopram

Simul taneous: chlorprothixene, clomipramine, clozapine, flupenthixol, haloperidol, levo-

mepromazine, perphenazine, zuclopenthixol

Noninterfer ing: benzodiazepines

Interfering: desmethyllevomepromazine

KEY WORDS

serum

REFERENCE

Olesen, O.V.; Linnet, K. Simplified high-performance liquid chromatographic method for the determi-

nation of citalopram and desmethylcitalopram in serum without interference from commonly used

psychotropic drugs and their metabolites.

J.Chromatogr.B

1996,

675

8 3 - 8 8

SAMPLE

Matrix: blood

Sample preparat ion:

1 mL Plasm a + doxepin + N aOH + h exa ne : isoamyl alcohol 98 :2,

extract. R emove the organic pha se and add it to 0.03% phosphoric acid, extract, inject an

aliquot of the aqueous phase.

HPLC VARIABLES

Gu ard column: C18

Column: 100 X 8 10 |xm Resolve C8 (Waters)

Mobile phase:

MeC N: M eOH: 56 mM am monium acetate :1 M ammonium hydroxide 100:

10:4.5:2.6

Nortriptyline

Molecular formula: C

9

H

2

N

Molecular weight: 263.4

CAS Registry No.: 72-69-5, 894-71-3 (HCI)


9/36

Flow rate: 2.5

Detector: UV

220

CHROMATOGRAM

Retent ion t ime: 31

Internal s tandard:

doxepin (11.6)

OTHER SUBSTANCES

Extracted:

amitriptyline, fluoxetine, norfluoxetine

KEY WORDS

plasma

REFERENCE

el-Yazigi, A.; Chaleby, K.; Gad, A.; Raines, D.A. Steady-state kinetics of fluoxetine and amitriptyline in

patien ts treated with a combination of these drug s as compared with those treated w ith amitriptyline

alone. J.CHn.Pharmacol. 1995, 35 1 7 - 2 1

SAMPLE

Matrix:

blood

Sample preparat ion: 200

\x L

Plas m a or seru m + 100 jxL 2 M pH 10.6 Tris buffer + 200

IJiL M TBE, vorte x for 30 s, centrifuge at 9950 g for 3 min , inject a 50 JJLL aliquot of the

organic layer.

HPLCVARIABLES

Guard column:

10 X 4.6 SCX-10C5 (Hichrom)

Column: 150 X 4.6 S pher isorb S5 SCX (sulfopropyl-bonded silica) cation exchang e

Mobile phase:

35 mM ammonium perchlorate in MeOH adjusted to an apparent pH of 6.7

with 100 mM NaOH in MeOH

Fl ow rate: 1.5


Detector: UV 215

CHROMATOGRAM

Retent ion t ime: 9

Internal s tandard: nortriptyline hydrochloride

OTHER SUBSTANCES

Extracted:

clozapine

Simul taneous: amitriptyline, chlorpromazine, clomipramine, dothiepin, doxepin, fluoxe-

t ine,

fluphenazine, fluvoxamine, haloperidol, imipramine, maprotiline, mianserin, nor-

clomipramine, nordothiepin, nordoxepin, norfluoxetine, nortriptyline, paroxetine, remox-

ipride, sulpride, thioridazine, trazodone

Noninterfering:

carbam azepine, clonazepam, diazepam , flunitrazepam, lorazepam, nordi-

azepam, theophylline

Interfering:

sertraline

KEYWORDS

plasma; serum; nortriptyline is IS

REFERENCE

McCarthy, P.T.; Hughes, S.; Paton, C. Measurement of clozapine and norclozapine in plasma/serum by

high-performance liquid chromatography with ultraviolet detection. Biomed.Chromatogr. 1995, 9

3 6 - 4 1


10/36

SAMPLE

Matrix: blood


2 mL Whole blood or pla sm a + 2 mL buffer + 5 mL chloroform:

isopropanol: n-he ptane 60 :1 4:2 6, sh ake g ently horizontally for 10 min, centrifuge a t 2800

g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum

at 45°, reconstitute the residue in 100 fxL 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% amm onia solution.)

HPLCVARIABLES

Column:

300 X 3.9 4

\xm

NovaPack C18

Mobile phase: M eO H :TH F:b uffer 65 :5 :3 0 (Buffer w as 0.68 g/L (10 mM (sic)) KH

2

PO

4

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.)

Colum n tem peratur e: 30

Flow rate: 0.8


Detector: UV 239

CHROMATOGRAM

Retent ion t ime: 9.28

Limit of detect ion:


11/36

Interfering:

amitriptyline, daunorubicin, diclofenac, etodolac, fluoxetine, indomethacin,

maprotiline, mefloquine, tioclomarol, trimipramine, tropatenine

KEYWORDS

whole blood; plasma

REFERENCE

Tracqui, A.; Kintz, P.; Mangin, P. Systematic toxicological analysis using HPLC/DAD.

J.Forensic ScL

1995, 40 254-262

SAMPLE

Matrix: blood


1 mL Seru m + 500 |xL 250 |xg/mL pro tript ylin e hydrochloride + 1

mL 500 mM NaOH + 4 mL toluene : n-hex ane: 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 to

dryn ess un der a stre am of air a t 40°, recon stitute th e residu e in 200 |xL MeOH , inject a

50 |xL aliquot.

HPLCVARIABLES

Column: 150 X 4.7 5 |xm Supelcosil LC-PCN cyanopropyl

Mobile phase:

MeC N: M eOH: 10 mM pH 7.2 potassium phosp hate buffer 60 :15 :25 (Pre-

pare buffer by mixing 194 mL 1.36 g/L KH

2

PO

4

with 274 mL 1.74 g/L K

2

HPO

4

.)

Flow rate: 2

Inject ion vo lume: 50

Detector: UV

254

CHROMATOGRAM

Retent ion t ime:

6.95

Internal s tandard:

protriptyline (8.1)

OTHER SUBSTANCES

Extracted:

amitriptyline, cyclobenzaprine

Interfering: norcycyclobenzaprine

KEYWORDS

serum

REFERENCE

Wong, E.C .C ; Koenig, J.; Turk, J. P oten tial interference of cyclobenzaprine a nd norcyclobenzaprine with

HPLC measurement of amitriptyline and nortriptyline: resolution by GC-MS analysis. J.Anal.

ToxicoL 1995, 19 218-224

SAMPLE

Matrix: blood

Sample preparat ion: Automated SPE by ASPEC system . Condition a C18 Clean-Up SPE

cartridge (CEC

18111,

Worldwide Monitoring) with 2 mL MeOH then 2 mL water. 1 mL

Pla sm a + 1 mL 400 ng/mL protripty line in water, vortex, add to th e SPE cartridg e, wash

with 3 mL water, wash with 3 mL 750 mL/L methanol. Elute with three aliquots of 300

IxL 0.1 M ammonium acetate in MeOH. Add 0.5 mL 0.5 M NaOH and 4 mL 50 mL/L

isopropanol in heptane to eluate, mix thoroughly. Allow 5 min for phase separation. Re-

move upper heptane phase and add it to 300 JJLL 0.1 M phosphoric acid (pH 2.5), mix,

separate, inject a 100 |xL aliquot of the aqueous phase.

HPLCVARIABLES

Guard co lumn :

LC-8-DB (Supelco)

Column: 150 x 4.6 LC-8-DB (Supelco)


12/36

Mobile phase: M eCN : buffer 35 :65 (Buffer was 10 mL/L trieth ylam ine in w ater adjusted

to pH 5.5 with glacial acetic acid.)

Flow rate: 2

Inject ion vo lum e: 100

D etecto r: UV 228

CHROMATOGRAM

Reten tion time: 4.8

Internal s tandard: protriptyline (4)

OTHER SUBSTANCES

Extracted:

acetazolamide, amitriptyline, chlordiazepoxide, chlorimipramine, chlorproma-

zine,

desipramine, dextromethorphan, diazepam, diphenhydramine, doxepin, encainide,

fentanyl, flecainide, fluoxetine, fturazepam, haloperidol, hydroxyethylflurazepam, ibupro-

fen, imipramine, lidocaine, maprotiline, methadone, methaqualone, mexiletine, midazo-

lam, norchlorimipramine, nordiazepam, nordoxepin, norfluoxetine, norverapamil, pentaz-

ocine, promazine, propafenone, propoxyphene, propranolol, protriptyline, quinidine,

temazepam, trazodone, trimipramine, verapamil

Noninterfer ing:

acetaminophen, acetylmorphine, amiodarone, amob arbital, 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, warfarin

Interfering: imipramine, maprotiline, verapamil

KEYWORDS

plasma; SPE

REFERENCE

Nichols, J.H.; Charlson, J.R.; Lawson, G.M. Automated HPLC assay of fluoxetine and norfluoxetine in

serum. Clin.Chem. 1994, 40 1312-1316

SAMPLE

Matrix:

blood


Condition a 1 mL BondElut C18 SPE cartridge with 1 mL 1 M HCl,

1 mL MeOH, 1 mL water, a nd 1 mL 1% potass ium carbon ate. 700 \xL Se rum + 50 |xL 5

(xg/mL trim ipra m ine in 5% potass ium bicarbona te + 700 |xL MeCN , vortex, centrifuge at

1500 g for 5 min, add supernatant to SPE cartridge (at ca. 1 mL/min). Wash with 2 mL

wa ter and 1 mL M eCN, elute w ith 250 |xL M eOH : 35% perchloric acid 20 :1 by g ravity

(10 min) then centrifuge for 20 s to remove rest of eluant, inject a 50

|JLL

aliquot of the

eluate.

HPLCVARIABLES

Guard co lumn: 15 mm 7 |jim Brownlee RP-8

Column:

150 X 4.6 5 jxm Ultrasphere Octyl

Mobile phase: MeC N: water 37.5:62.5 containing 0.5 g/L tetrameth ylam mo nium perchlo-

rate and 0.5 mL/L 7% perchloric acid

F lo w rate : 1.5


Detector: UV

215


13/36

CHROMATOGRAM

Retention time: 7.4

Internal standard: trimipramine (9.6)

Lim it of qua nt ita tio n: 5 ng/mL

OTHER SUBSTANCES

Extracted: amitriptyline, clomipramine, desipramine, doxepin, fluoxetine, fluvoxamine,

maprotiline, protriptyline

In te r fe r ing : imip ramine

KEYWORDS

serum; SPE

REFERENCE

Gupta, R.N. An improved solid phase extraction procedure for the determination of antidepressants in

serum by column liquid chromatography. J.Liq.Chromatogr. 1993, 16 2751-2765

SAMPLE

Matrix: blood

S a m p l e p r e p a r a t i o n : 500 \xL Ser um + 250 |xL 100 mM lau ryl sulfate, cen trifuge a t 2500

g for 8 min, inject a 250 JULL aliquot of th e s up ern ata nt onto column A with mobile phas e

A, elute with mobile phase A for 6 min, backflush contents of column A onto column B

with m obile phase B for 4 min. E lute column B w ith mobile phas e B for 6 m in and conduct

analysis. W hen not in use flush column A with m obile ph ase A. Every eight injections

backflush column A with M eCN: wa ter 70:30.

HPLC VARIABLES

Column: A Guard-Pak 10 |xm Resolve CN (Waters); B 150 X 3 7 jxm Separon SGX CN

(Tessek)

M ob ile p h a s e : A MeC N: water 3:97; B MeCN : buffer 26:74 (Buffer was 50 mM phosphoric

acid, 50 mM ammonium phosphate, and 28 mM diethylamine, pH 2.55.)

F l o w r a t e : 1

In jec t ion vo lume : 250

D ete c to r : UV 210


Retention time: 10

Limit of detec tio n: 15-20 ng/mL

OTHER SUBSTANCES

Extracted: amitriptyline

KEYWORDS

serum; column-switching

REFERENCE

Dolezalova, M. On-line solid-phase extraction and high-performance liquid chromatographic determi-

nation of nortriptyline and amitriptyline in serum. J.Chromatogr.

1992,

579 291-297

SAMPLE

Matrix: blood

Sample preparation: Add 10 |xL 20 |xg/mL oxaprotiline in MeOH to 990 JJLL plasma or

serum . Inject 100 jxL plas m a or se rum onto column A with m obile phase A and e lute to

was te, after 15 min elute column A onto column B with mobile pha se B for 2 min. Remove


14/36

column A from circuit an d re-eq uilibrate it with mobile pha se A for 5 min. Ch rom atograp h

on column B with mobile phase B.

HPLCVARIABLES

Column: A 20 X 4.6 10 |xm Hypersil MOS C8; B 20 X 4.6 5 |xm Hypersil CPS CN + 250

x 4.6 5 |xm Nucleosil 100 CN

Mobile phase:

A MeOH: water 5:95; B MeOH : MeCN: 10 mM pH 6.8 potassium phosphate

buffer 188:578:235

Flow ra te: 1.5


Detector: UV

214

CHROMATOGRAM


Internal s tandard: oxaprotiline (9.5)

Limit of detect ion: 20 ng/mL

OTHER SUBSTANCES

Extracted: amitriptyline, clomipramine, clozapine, desipramine, doxepin, fluoxetine, flu-

voxamine, imipramine, maprotiline, metoclopramide, norfluoxetine

Noninterfer ing: carbamazepine, chlordiazepoxide, clobazam, diazepam, flurazepam, flus-

pirilene, haloperidol, lorazepam, nitrazepam, nordiazepam, oxazepam, perazine, pimo-

zide, spiroperidol, trifluperidol

KEYWORDS

plasma; serum; column-switching

REFERENCE

Hartter, S.; Wetzel, H.; Hiemke, C. Automated determination of fluvoxamine in plasma by column-

switching high-performance liquid chromatography. Clin.Chem. 1992, 38 2082-2086

SAMPLE

Matrix:

blood

Sample preparat ion: For each 1 mL plasm a or serum add 10 ^L 14 jxg/mL trimi pra m ine

in MeOH. Inject serum or plasm a directly onto column A with m obile phase A, elute w ith

mobile phase A to waste. After 15 min elute column A onto column B (foreflush) with

mobile pha se B . After 2 min rem ove column A from th e circuit, elute column B with m obile

ph ase B , mon itor the effluent from column B. Re-equilibrate column A with m obile ph ase

A.

HPLCVARIABLES

Column: A 20 X 4.6 10 jxm Hypersil MOS C8; B 20 X 4.6 5 |xm Hypersil CPS CN + 250

x 4.6 5 [xm Nucleosil 100 CN

Mobile phase: A M eOH : w ater 5:9 5; B M eCN : M eOH : buffer 578 :18 8:23 5 (Buffer wa s 10

mM K

2

HPO

4

adjusted to pH 6.8 w ith 85% phosphoric acid.)

Flow rate: 1.5

Inject ion vo lume:

100

Detector: UV

214

CHROMATOGRAM


Internal s tandard:

trimipramine (6.5)

Limit of detect ion: 5-10 ng/mL

OTHER SUBSTANCES

Extracted: metabolites, amitriptyline, clomipramine, desipramine, doxepin, fluvoxamine,

imipramine, maprotiline


15/36

Noninterfer ing: chlordiazepoxide, clobazam, clozapine, diazepam, flurazepam, fluspiri-

lene,

haloperidol, nitrazepam, oxazepam, perazine, pimozide, spiroperidol, trifluperidol

KEYWORDS

plasma; serum; column-switching; LOD can be lowered to 1 ng/mL with 3 injections onto

column A before switch ing

REFERENCE

Hartter, S.; Hiemke, C. Column switching and high-performance liquid chromatography in the analysis

of amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum.

J.Chromatogr.

1992,

578

273-282

SAMPLE

Matrix: blood


1 mL Pla sm a + 100 |xL 1 M pH 7.6 K

2

HPO

4

, vortex for 5 s, add 6

mL ethyl acetate, vortex for 1 min, centrifuge at 1900 g for 5 min. Remove the organic

layer and e vapo rate it to dryness und er a st rea m of nitrogen at 45°, reco nstitu te th e

residue in 250 fxL 40 mM sodium bicarbonate, add 20 jxL 10 mg/mL dansyl chloride in

acetone, add 750 |xL acetone, vortex for 30 s, let stand at room temperature at 22° for 15

min. Ev apora te und er a strea m of nitrogen at 45°, reco nstitu te in 1 mL mobile phase,

vortex for 1 min, centrifuge at 1900 g for 10 min, inject a 100 |xL aliquot of the

superna tant .

HPLCVARIABLES

Guard co lumn :

15 X 3.2 7 jxm NewGuard RP-8 (Brownlee)

Column:

250 X 4.6 5 jxm Supelcosil LC-18-DB

Mobile phase:

MeC N: 10 mM pH 7.2 potassium p hosph ate 85:1 5

Flow ra te: 1.5

Inject ion vo lum e:

100

Detector: F (wavelengths not given)

CHROMATOGRAM

Retent ion t ime: 9 .7

Internal s tandard: nortriptyline

OTHER SUBSTANCES

Extracted: fluvoxamine

Simul taneous: desipramine

KEYWORDS

plasma; derivatization; nortriptyline is IS

REFERENCE

Pullen, R.H.; Fatmi, A.A. Determination of fluvoxamine in human plasma by high-performance liquid

chromatography with fluorescence detection. J.Chromatogr. 1992, 574 101-107

SAMPLE

Matrix:

blood


1 mL Pl asm a + 1 mL 0.6 M pH 9.8 carb ona te buffer + 40 |JLL 5

|xg/mL maprotiline in 10 mM HCl + 5 mL 200 g/L ethyl acetate in n-heptane, mix by

rocking for 10 min, centrifuge at 1500 g for 10 min. Remove organic layer and add it to

150 |xL 100 mM HCl, mix 10 min, centrifuge at 1500 g for 10 min. Discard organic layer

and evapo rate aqu eous layer a t 45° in a vacuum centrifuge for 1 h. Take up resid ue in

50 jxL 1 M pH 10.3 carb on ate buffer an d 25 |JLL 10 mg/mL dan syl chloride in M eCN,

vortex, allow to react at room temperature for 45 min, evaporate at 45° in a vacuum


16/36

centrifuge for 20 min, reconstitute in 125 |JLL M eCN -.water 75 :2 5, vortex , centrifuge for

3-5 min, inject a 25-40 |xL aliquot.

HPLCVARIABLES

Column: 250 X 4.6 5 jim Supelcosil LC-18

Mobile phase:

MeCN : 25 mM KH

2

PO

4

75:25 containing 500 |xL/L orthophosphoric acid and

600 fxL/L n-butylamine

Flow rate: 2

Inject ion volume:

25-40

Detector: F ex 235 em 470 (cut-off)

CHROMATOGRAM

Retent ion t ime:

14.18

Internal s tandard:

maprotiline (12.8)

OTHER SUBSTANCES

Simul taneous:

amoxapine, clovoxamine, desipramine, fenfiuramine, fluoxetine, fluvoxam-

ine,

norfluoxetine, propranolol, protriptyline, sertraline

Noninterfering: amitriptyline, atenolol, bupropion, carbamazepine, chlordiazepoxide, ci-

talopram, clomipramine, clozapine, cyclobenzaprine, doxepin, imipramine, loxapine, me-

toprolol, mianserin, moclobemide, nomifensine, pindolol, thioridazine, tranylcypromine,

trazodone, trimipramine

KEYWORDS

plasma

REFERENCE

Suckow, R.F.; Zha ng, M .F.; Cooper, T.B. Sen sitive and selective liquid -chrom atogra phic assa y of fluoxe-

tine and norfluoxetine in plasma with fluorescence detection after precolumn derivatization.

Clin.Chem. 1992, 38 1756-1761

SAMPLE

Matrix:

blood

Sample preparat ion: 1 mL Seru m + 3 |xL 20 ng/mL clobazam + 1 mL sat ur ate d sodium

borate (adjusted to pH 11 with 6 M NaOH) + 5 mL n-hex ane, mix 2 m in, centrifuge at

3000 g for 10 min. Remove organic phase and evaporate to dryness under a stream of

helium at 30°. Reconstitute in 20 |xL mobile phase, inject a 10 |xL aliquot.

HPLCVARIABLES

Guard column:

20 mm long Pelliguard LC-8 40 |xm (Supelco)

Column: 150 X 4.6 C8 5 \xm (Supelco)

Mobile phase: M eCN : buffer 50:5 0 (Buffer was 1.2 m L butylam ine in 1 L 10 mM N aH

2

PO

4

,

pH adjusted to 3 with phosphoric acid.)

Flow rate: 1


Detector: UV 254

CHROMATOGRAM

Retent ion t ime:

k' 2.460

Internal s tandard: clobazam (k' 1.344)

Limit of detect ion: 10 ng/m L

OTHER SUBSTANCES

Extracted: amitriptyline, clomipramine, desipramine, imipramine

Simul taneous:

alprazolam, clonazepam, diazepam, flunitrazepam, haloperidol, lorazepam,

maprotiline, nitrazepam, triazolam


17/36

KEYWORDS

serum

REFERENCE

Segatti, M.P.; Nisi, G.; Grossi, F.; Mangiarotti, M.; Lucarelli, C. Rapid and simple high-performance

liquid chromatographic determination of tricyclic antidepressants for routine and emergency serum

analysis. J.Chromatogr. 1991, 536 3 1 9 - 3 2 5

SAMPLE

Matrix:

blood


Inject 200 |xL serum onto column A and elute with mobile phase A

for 10 min the n back-flush column A onto column B with mobile ph ase B for 4 min. El ute

column B with m obile ph ase B and m onitor th e effluent. Remove column A from circuit

and w ash w ith MeC N: w ater 60:4 0 for 6 min the n w ith mobile phas e A for 10 min.

HPLCVARIABLES

Column: A 40 X 4 TSKprecolumn PW (Tosoh); B 150 X 4 TSKgel ODS-80TM (Tosoh)

Mobile phase:

A 50 mM pH 7.5 potassium phosp hate; B MeCN: 100 mM pH 2.7 potassium

pho sph ate 32.5:67 .5, containin g 0.2 g/L sodium 1-heptanesulfonate

Flow rate: 1

Inject ion volume:

200

Detector: UV 210

CHROMATOGRAM



10 ng/mL

OTHER SUBSTANCES

Extracted: amitriptyline, amoxapine, clomipramine, doxepin, desipramine, maprotiline,

tr imipramine

Interfering: imipramine

KEYWORDS

serum; column-switching; use gradient to determine metabolites

REFERENCE

Matsumoto, K.; Kanba, S.; Kubo, H.; Yagi, G.; Iri, H.; Yuki, H. Automated determination of drugs in

serum by column-switching high-performance liquid chromatography. IV. Sep aration of tricyclic and

tetracyclic antidepressants and their metabolites.

C lin.Chem.

1989,

35

4 5 3 - 4 5 6

SAMPLE

Matrix:

blood


1 mL P las m a + 50 [xL M eO H: w ate r 5 0:5 0 + 50 |xL 10 |xg/mL des-

methyldoxepin in MeO H: wa ter 50 :50 , mix, inject a 250 |xL aliquot of thi s m ixture onto

column A and elute to waste with mobile phase A. After 1.5 min backflush the contents

of column A onto column B with mobile phase B, monitor the effluent from column B.

HPLCVARIABLES

Column: A 37-50 |xm 10 X 1.5 Co rasil RP C18; B 100 X 4 Tech sphere 3CN (HPLC

Technology)

Mobile phase:

A water; B M eCN: 50 mM ace tate buffer 60:40 , pH 7

F lo w r ate : A 0.8; B 0.9

Inject ion vo lume : 250

Detector: UV

215


18/36

CHROMATOGRAM

Retention t ime: 10.0

Internal s tandard: desmethyldoxepin (8.5)


5-10 ng/mL

OTHER SUBSTANCES

Extracted:

metabolites, amitriptyline

Simul taneous:

chloripramine, clomipramine, desipramine, doxepin, imipramine, protrip-

tyline

Noninterfering: tranylcypromine

Interfering:

cianopramine, trimipramine

KEYWORDS

column-switching; plasma

REFERENCE

Dadgar, D.; Power, A. Applications of column-switching technique in biopharmaceutical analysis. I.

High-performance liquid chromatographic determination of amitriptyline and its metabolites in hu-

man plasma. J.Chromatogr. 1987, 416 9 9 - 1 0 9

SAMPLE

Matrix: blood


Condition a Bond Elut C-18 SPE cartridge twice with MeOH and

twice with water. 500 JULL Serum + 50 JULL 1 |xg/mL N-propionylprocainamide in 2.5 mM

HCl, add to the SPE cartridge, wash with 2 volumes water, wash with 2 volumes 0.1 M

acetic acid, was h with 1 volume M eOH : 2.5 mM HCl 10:90 . Add 200 jxL 10 mM acetic

acid and 5 mM diethylam ine in MeOH to column, let stan d 1 m in, elute un der vacuu m ,

repeat, evaporate eluents to dryness under nitrogen at room temperature, reconstitute in

100 |xL mobile phase, inject a 40 |JLL aliquot.

HPLCVARIABLES

Guard column: Pelliguard LC-CN (Supelco)

Column:

150 X 4.6 5

[xm

Supelcosil LC-PC N

Mobile phase: M eCN :MeO H: 10 mM pH 7.0 phospha te buffer 58 :14 :28

F low rate: 1.2


D etector : UV 254

CHROMATOGRAM


Internal s tandard: N-propionylprocainamide (6)

Limit of quantitation:

25 ng/mL

OTHER SUBSTANCES

Extracted: amitriptyline, desipramine, doxepin, imipramine, protriptyline, trimipramine

Simul taneous: atropine, butalbital, chlorpromazine, maprotiline, methadone, norpropoxy-

phene, phenylpropanolamine, procainamide, prochlorperazine, promethazine, proprano-

lol, quinidine, trifluoperazine, trimeprazine

Noninterfer ing: acetamin ophen , allopurinol, amikacin, amoxap ine, am ytal, brety lium , caf-

feine, carbamazepine, carisoprodol, chloramphenicol, chlordiazepoxide, chlorpropamide,

clonazepam, codeine, diazepam, disopyramide, droperidol, ethinamate, ethinamate, etho-

suximide, fluphenazine, flurazepam, furosemide, gentamicin, haloperidol, hydrochlo-

rothiazide, hydroxyzine, ibuprofen, kanamycin, lidocaine, loxapine, meperidine, mepho-

barbital, meprobamate, methaqualone, methotrexate, morphine, nafcillin, naloxone,

neomycin, perphenazine, phenacetin, phenobarbital, phenytoin, prazepam, primidone,

procaine, propoxyphene, reserpine, salicylamide, salicylic acid, secobarbital, spironolac-

tone, theophylline, thiopental, thioridazine, tobramycin, valproic acid, verapamil


19/36

KEYWORDS

serum; SPE

REFERENCE

Lin, W.-N.; Frade, RD. Simultaneous quantitation of eight tricyclic antidepressants in serum by high-

performance liquid chromatography. Ther.Drug Monit 1987, 9 4 4 8 - 4 5 5

SAMPLE

Matrix: blood

Sample preparation:

500 |xL Serum + 250 |xL di-iso-propyl ether: n-butyl alcohol 7:3 con-

taining 800 ng/mL minaprine, centrifuge 2 min, shake, centrifuge 5 min, inject 50 |xL

aliquot of top organic layer.

HPLCVARIABLES

Guard column: 30 X 4.6 5 |xm Brownlee cyano spheri-5

Column: 250 X 4.6 5 |xm Altex ultrasphere cyano

Mobile phase:

MeCN: THF: water: 2 M ammonium formate (pH 4.0) 700:100:195:5


Flow rate: 1.5


Detector: UV 240

CHROMATOGRAM

Retention time: 6.5

Internal standard:

minaprine (5.5)

Limit of detection: 20 ng/mL

OTHER SUBSTANCES

Simultaneous: amitriptyline, diltiazem

Also analyzed: amiodarone, clomipramine, desipramine, haloperidol, imipramine, propa-

fenone, verapamil

KEYWORDS

serum

REFERENCE

Mazzi, G. Simple and practical high-performance liquid chromatographic assay of some tricyclic drugs,

haloperidol, diltiazem, verapamil, propafenone, and amiodarone. Chrom atographia 1987, 24 313-316

SAMPLE

Matrix: blood

Sample preparation: 1 mL Serum + 1 mL 450 mM NaOH + 5 mL hexane: isopropanol

95:5,

shake for 5 min, centrifuge. Remove 4 mL of the organic layer and add it to 50 |JLL

200 mM HCl, shake for 2 min, centrifuge. Inject a 20 |xL aliquot of the aqueous layer.

HPLCVARIABLES

Column: 100 X 3 octyl CP-tm-Spher C8 glass column (Chrompack)

Mobile phase: MeCN: 500 mM NaH

2

PO

4

35:65 adjusted to pH 2.2 with phosphoric acid

Flow rate: 0.8


Detector: UV 210

CHROMATOGRAM

Retention time: 4.5



20/36

OTHER SUBSTANCES

Sim ult an eo us : amitriptyline, doxepin

KEYWORDS

serum

REFERENCE

Van Damm e, M.; Molle, L.; Abi Khalil, F. Useful samp le hand lings for reversed p has e high performance

liquid chromatography in emergency toxicology. J.Toxicol.Clin.ToxicoL 1985, 23 5 8 9-6 1 4

SAMPLE

Matrix: blood

Sample preparation: 2 mL Serum or plasma + 100 |JLL 1 |xg/mL IS in water + 0.5 mL

water, vortex, extract with 10 mL toluene: isoamyl alcohol 99 :1 for 10 min on a rotator,

centrifuge for 5 min. Remove upper organic layer, evaporate under a stream of nitrogen

at 37°, take up in 150 |xL mobile phase, vortex for 2 min, add 0.5 mL hexane, vortex

briefly, centrifuge for 5 min, discard upper hexane layer, inject a 100 jxL aliquot of the

lower layer.

HPLCVARIABLES

Colum n: 250 X 4 Bio-Sil ODS-10 (Bio-Rad)

M obile phase : MeCN :pH 4.5 50 mM phosphate buffer 30:70 (Buffer was 6.9 g KH

2

PO

4

in

1 L adjusted to pH 4.5 with orthophosphoric acid.)


Flow rate: 2.5


D etecto r: UV 202


Retention time: 8.4

In te rn al s ta nd ar d: U-31485 (6.9)

Lim it of de tec tio n: 1 ng/mL

OTHER SUBSTANCES

Extracted: desipramine, protriptyline

Noninterfering: N-acetylprocainamide, amitriptyline, caffeine, chlordiazepoxide, chlor-

promazine, diazepam, flurazepam, lorazepam, oxazepam, prazepam, procainamide, pro-

pranolol, thioridazine

In te rf er in g: alprazolam, imipramine, triazolam

KEYWORDS

serum; plasma

REFERENCE

McCormick, S.R.; Nielsen, J.; Jatlow, P. Quantification of alprazolam in serum or plasma by liquid

chromatography. Clin.Chem. 1984, 30 1652-1655

SAMPLE

Matrix: blood

Sam ple pr ep ar at io n : 500 jxL Plasma + 37 |JLL 2 |xg/mL IS in MeOH + 500 ^xL pH 10

borate buffer + 1 .5 mL hexane: isoamyl alcohol 95 :5, shake for 10 min. Evaporate the

organic layer to dryness under a stream of nitrogen, reconstitute in 100 IJLL MeOH, inject

a 50 |xL aliquot. (The borate buffer was prepared as follows. Prepare a solution of 61.8 g

boric acid and 74.6 g KCl in 1 L water. Add 630 mL of this solution to 370 mL 106 g/L

sodium carbonate solution. Adjust pH to 10.0 with 6 M NaOH and store at 35-37°.)


21/36

HPLCVARIABLES

Column: 250 X 4.6 Zorbax SiI

Mobile phase:

M eOH: amm onium hydroxide 99 8:2

F lo w rat e: 1.5


Detector: UV 254

CHROMATOGRAM

Retent ion t ime: 9

Internal s tandard: N-desmethylclomipramine hydrochloride (10)

Limit of quant i tat ion:

20 ng/mL

OTHER SUBSTANCES

Extracted: metabolites, amitriptyline, desipramine, 2-hydroxydesipramine, 2-hydroxyimi-

pramine, imipramine

Also analyzed:

clomipramine, desmethylclomipramine, desmethyldoxepin, doxepin, ma-

protiline, protriptyline

Noninterfer ing:

chlordiazepoxide, diazepam, flurazepam, oxazepam, thioridazine

KEYWORDS

plasma

REFERENCE

Sutfin, T.A.; D'Ambrosio, R.; Jusko, W.J. Liquid-chromatographic determination of eight tri- and tetra-

cyclic antidepressants and their major active metabolites. Clin.Chem. 1984, 30 4 7 1 - 4 7 4

SAMPLE

Matrix: blood


Condition a Bond-Elut C18 column with 2 volumes MeOH then 2

volumes water. Add 1 mL serum the n 200 |xL 700 ng/mL pro mazine in Me OH : 0.1 M HCl

13:87 to each column, wash with 2 volumes water, wash with 2 volumes 0.1 M acetic

acid, wash with MeOH/water, add 200 |xL 10 mM ammonium acetate in MeOH, wait for

30 s, elute with vacuum, repeat elution process two more times. Combine eluates and

evapo rate them to dryne ss a t 56-8° un der compressed air. Re constitute w ith 200 |xL mo-

bile phase, vortex 10 s, inject 75-100 |xL aliquot. (MeOH/water was 500 mL MeOH-.water

65 :35 plus 25 jxL concentrated HCl.)

HPLC VARIABLES

Column: 250 X 4.6 5 fim Supelco

Mobile phase:

1 gal EtOH + 77 mL MeCN + 1.9 mL t-butylamine (EtOH : MeC N: t-buty-

lamine 98:2:0.05)

Flow rate: 2

Inject ion volume: 75-100

Detector: UV

254

CHROMATOGRAM


Internal s tandard:

promazine (5.2)


OTHER SUBSTANCES

Extracted: amitriptyline, desipramine, desmethyldoxepin, doxepin, imipramine, protrip-

tyline

Simul taneous: N-acetylprocainamide, amoxapine, amphetamine, buprion, chlordiazepox-

ide,

chlorimipramine, chlorpheniramine, chlorpromazine, cocaine, codeine, demoxepam,

desmethylchlordiazepoxide, desmethyldisopyramide, dextropropoxyphene, diazepam, di-


22/36

sopyramide, fluphenazine, hydroxyamoxapine, 2-hydroxydesipramine, 2-hydroxyimipra-

mine, 10-hydroxyno rtriptyline, iminostilbene, iprindole, loxepin, ma protiline, mep eridine,

methadone, mianserin, morphine, norzimeldine, oxapam, oxaprotiline, perphenazine,

phenteramine, procainamide, prolixin, promethazine, propoxyphene, pyrilamine, qui-

nidine, thioridazine, trifluoperazine, trifluopromazine, trimeprazine, trimipramine,

zimeldine

Noninterfer ing:

thiopropazine

Interfering: prochlorperazine

KEYWORDS

serum; normal phase

REFERENCE

Beierle, RA.; Hubbard, R.W. Liquid chromatographic separation of antidepressant drugs: I. Tricyclics.

Ther.Drug Monit 1983, 5, 27 9-29 2

SAMPLE

Matrix:

blood


2 mL Plasm a + 100 JULL 1 |mg/mL loxapine in isopr opan ol: diethy lam -

ine 99.9 :0.1 + 250 |xL 25% potas sium carbon ate containing 0.1% diethylam ine + 5 mL

he xa ne : isoamyl alcohol 9 7 :3 , vortex for 30 s, centrifuge at 500 g for 3 m in. Remove t h e

organic layer and add it to 100 |xL 250 mM HCl, vortex for 30 s, inject a 50 |JLL aliquot

of the aqueous phase.

HPLCVARIABLES

Guard co lumn :

50 X 4.6 40 (xm C8 (Supelco)

Column:

250 X 4.6 5 jxm Supelcosil C8

Mobile phase:

MeCN:water:diethylamine:85% phosphoric acid 53.3:45.1:1:0.4, pH ad-

justed to 7.2 with NaOH or phosphoric acid

Flow rate: 2

Inject ion vo lume : 50

Detector: UV

254

CHROMATOGRAM

R ete nt ion t ime : k 3.45

Internal s tandard:

loxapine (k' 7.18)

Limit of detect ion: 2.5 ng/mL

OTHER SUBSTANCES

Extracted:

amitriptyline, chlordiazepoxide, chlorpromazine, desipramine, desmethldiaze-

pam, desmethylchlordiazepoxide, desmethyldoxepin, diazepam, doxepin, fluphenazine,

imipramine, oxazepam

Noninterfer ing:

molindone, perp hen azine , trifluoperazine

Interfering: haloperidol, thiothixene

KEYWORDS

plasma

REFERENCE

Kiel, J.S.; Abramson, R.K.; Morgan, S.L.; Voris, J.C . A rapid high performance liquid chrom atograph ic

method for the simultaneou s m easurem ent of six tricyclic antide pressan ts. J.Liq.Chromatogr. 1983,

6 2761-2773

SAMPLE

Matrix:

blood


23/36

Sample preparat ion: 10 mL Pla sm a or whole blood + 1 mL 1 M NaO H, extrac t twice

with 10 mL hexane for 30 min. Remove the organic layers and evaporate them to dryness

under a stream of nitrogen, reconstitute the residue in 1 mL 100 mM HCl, add 5 mL

chloroform, vortex for 1 min, centrifuge. Remove a 4.5 mL aliquot of the organic layer

and evapo rate it to dryne ss, recon stitute the residu e in 100 jxL mobile phas e, inject a 50

IxL aliquot. (It is implied, but not explicitly stated in the paper, that this extraction pro-

cedure works for this compound.)

HPLCVARIABLES

Column:

10 fxm Micropak CN (Varian)

Mobile phase:

MeCN: 20 mM am monium acetate 9 0:10

Flow rate: 2 .5


Detector: UV

254

CHROMATOGRAM

Retent ion t ime:

10.7


10 ng/mL

OTHER SUBSTANCES

Simul taneous: acetophenazine, amitriptyline, benztropine, butaperazine, carphenazine,

chlorpromazine, fluphenazine, haloperidol, imipramine, mesoridazine, orphenadrine, pi-

peracetazine, promazine, promethazine, thioridazine, thiothixene, trifluoperazine, triflu-

promazine, trihexyphenidyl, trimeprazine

KEYWORDS

plasma; whole blood

REFERENCE

Curry, S.H.; Brown, E .A.; Hu,

O.Y.-R;

Per rin, J.H . Liquid chromatog raphic a ssay of phen othiazine , thiox-

anth ene and butyrophenone neuroleptics and a ntihistam ines in blood and plasma w ith conventional

and radial compression columns and UV and electrochemical detection. J.Chromatogr. 1982, 231

361-376

SAMPLE

Matrix: blood


1 mL S eru m + 200 |xL 10 fxg/mL pro trip ty line in wa ter + 200 fxL

80 g/L N aHC O

3

+ 5 mL hexane, vortex for 15 s, centrifuge for 5 min. Remove the hexane

layer and evapo rate it in a strea m of nitrogen at 60°. Rec onstitute in 100 JULL mobile pha se,

vortex for 15 s, inject a 50 (JLL aliquot.

HPLCVARIABLES

Column:

300 X 4 10 |xm jjiBondapak CN

Mobile phase: M eCN :MeO H: 5 mM pho sphate buffer 60 :15 :25 , adjusted to pH 7.0

Flow rate: 2


Detector: UV

254

CHROMATOGRAM

Retent ion t ime:

7.10

Internal s tandard: protriptyline (12.20)


10 ng/mL

OTHER SUBSTANCES

Extracted: amitriptyline, chlorpromazine, desipramine, desmethyldoxepin, doxepin, imip-

ramine, maprotiline, procainamide, propoxyphene, propranolol, thioridazine, trimipra-

mine


24/36

Noninterfer ing:

acetam inophen , caffeine, chlordiazepoxide, diazepam , m ethaq ualon e, sal-

icylic acid, theophylline, trifluoperazine

Interfering:

disopyramide

KEYWORDS

serum

REFERENCE

Koteel, P.; Mullins, R.E.; Gadsden, R.H. Sample preparation and liquid-chromatographic analysis for

tricyclic antidepressants in serum. Clin.Chem.

1982,

28 462-466

SAMPLE

Matrix:

blood

Sample preparat ion: 2 mL Plasm a + 1600 ng clomipramine in MeOH + 2 mL 1 M NaOH

+ 5 mL h exa ne : isoamyl alcohol 9 9 :1 , sha ke m echanically for 15 min, centrifuge at 1686

g for 5 min. Remove the organic phase and add it to 200 |xL 0.05% orthophosphoric acid,

shake for 15 min, centrifuge for 5 min, inject a 50

JJLL

aliquot of the aqueous phase.

HPLCVARIABLES

Guard co lumn : ixBondapak/Porasil

Column: jxBondapak C18

Mobile phase:

MeCN:buffer 40:60 (Buffer was 13.68 g KH

2

PO

4

in 2 L water, adjusted to

pH 4.7 with dilute KOH.)


Flow rate: 2


50

Detector: UV

254

CHROMATOGRAM

Retention time: 4.5

Internal s tandard: clomipramine (7.5)


0.6 ng

OTHER SUBSTANCES

Extracted: amitriptyline, desipramine, imipramine

Simul taneous:

chlordiazepoxide, chlorpromazine, cimetidine, clomipramine, diazepam ,

doxepin, flurazepam, lorazepam, oxazepam, pentobarbital, perphenazine, phenobarbital,

phenytoin, prochlorperazine, propoxyphene, secobarbital, thioridazine, trifluoperazine

Noninterfering: acetaminophen, codeine, meperidine

KEYWORDS

plasma

REFERENCE

Wong,

S.H.Y.;

McCauley, T. Reversed phase high-performance liquid chromatographic analysis of tri-

cyclic antidepessants in plasma. J.Liq.Chromatogn

1981,

4 849-862

SAMPLE

Matrix:

blood, dialysate

Sample preparat ion: Adjust pH of serum samples to 7.4 and dialyze 3 mL serum against

5 mL dialysis buffer at 37° in PT FE cha m bers for 4 h. Inject 1 mL m obile pha se, 1 mL

water, 2 mL dialysate, 1 mL water, an d 1 mL MeC N: wa ter 5 0:50 onto column A. Elute

column A onto column B w ith m obile pha se for 30 s th en remove it from the circuit. E lute

column B with mobile phase and monitor the effluent. (Dialysis buffer was 3.998 g

Na

2

HPO

4

.2H

2

O + 0.775 g NaH

2

PO

4

+ 2.250 g NaCl + 0.055 g Hg(NO

3

)

2

in 1 L water, pH

was 7.4.)


25/36

HPLCVARIABLES

Column:

A 10 X 6 packed with 40 |xm ma teria l from a Bond Elu t cartridg e (cat. no. 620303);

B 100 X 4 3 iJtm Spherisorb ODS Superpac

Mobile phase: MeC N: 85% phosphoric acid: triethyla m ine: wa ter 49.55:0.225 :0.225:50

Flow rate:

0.65

Inject ion volume: 1000-2000

Detector: UV

238

CHROMATOGRAM

Retent ion t ime:

3.85

Limit of detection: 0.5 nM (5 mL sample)

OTHER SUBSTANCES

Extracted:

amitriptyline

Simul taneous: alprazolam, chlorpromazine, chlorprothixene, clomipramine, desclomipra-

mine, desmeth ylimip ram ine, diazepam , flunitrazepam, fluphenazine, haloperidol, imip-

ramine, levomepromazine, perphenazine, protriptyline, thioridazine, thioridazine sulfone,

trimipramine, zimeldine, zuclopenthixol

Noninterfering:

carbamazepine, clonazepam, lorazepam, nitrazepam, oxazepam, pheny-

toin

Interfering: maprotiline, promethazine, thioridazine sulfoxide

KEYWORDS

serum; column-switching

REFERENCE

Svensson, C; Nyberg, G.; Martensson, E. High-performance liquid chromatographic quantitation of am-

itriptyline and nortriptyline in dialysate from plasma or serum using on-line solid-phase extraction.

J.Chromatogr. 1988, 432 363-369

SAMPLE

Matrix: blood, gastric contents, tissue, urine


1 mL Blood, urin e, or gastric c ontents or 1 g tissu e homo genate +

500 |xL buffer + 8 mL n-h exa ne: ethyl ac etate 70:30 , mix on a rota ry m ixer for 10 min,

centrifuge at 3000 g for 8 min. Remove the organic layer and evaporate it to dryness

under a stream of nitrogen, reconstitute the residue in 100 |xL 12.5 mM NaOH in

M eOH : w ate r 5 0:5 0, inject a 50 |xL aliquot. (Buffer wa s 13.8 g po tassiu m carb on ate in

100 mL water, pH adjusted to 9.5 with concentrated HCl.)

HPLCVARIABLES

Guard column:

4 X 4 30 |xm LiChrocart Aluspher RP-select B (Merck)

Column: 125 X 4 5 fxm Alus phe r R P-select B (Merck)

Mobile phase:

Gradient. A was 12.5 mM NaOH in MeOH. B was 12.5 mM N aOH in w ater.

A:B 10:90 for 5 min, to 90:10 over 15 min, maintain at 90:10 for 5 min, return to initial

conditions over 1 min, re-equilibrate for 5 min.

Flow rate: 1

Inject ion volume:

50

Detector: UV

230;

UV

254

CHROMATOGRAM

Retention time: 19

OTHER SUBSTANCES

Extracted: alprenolol, amitriptylin e, bromazepa m, carba maz epine, chlordiazepoxide, chlor-

promazine, clonazepam, desipramine, diazepam, flunitrazepam, haloperidol, nitrendipine,

nordiazepam, pindolol, zolpidem


26/36

Also analyzed: acebutolol, acetaminophen, alprazolam, amphetamine, atenolol, betaxolol,

brotizolam, caffeine, camazepam, captopril, chloroquine, clobazam, clomipramine, cloth-

iapine, clotiazepam, cloxazolam, cocaine, codeine, diclofenac, dihydralazine, dihydrocod-

eine, dihydroergotamine, diphenhydramine, domperidone, doxepin, droperidol, ergota-

mine, ethyl loflazepate, fenethylline, fluoxetine, flupentixol, flurazepam, furosemide,

gliclazide, hydrochlorothiazide, hydroxyzine, ibuprofen, imipramine, ketazolam, loprazo-

lam, lorazepam, lormetazepam, maprotiline, medazepam, mepyramine, methadone,

methaqualone, methyldopa, methylphenidate, metoclopramide, metoprolol, mexiletine,

mianserin, midazolam, minoxidil, morphine, nadolol, nitrazepam, oxprenolol, papaverine,

pentazocine, phenprocoumon, phenylbutazone, pipamperone, piritramide, practolol, pra-

zepam, prazosin, promazine, promethazine, propoxyphene, propranolol, prothipendyl, qui-

nine, sotalol, sulpride, thioridazine, trazodone, triazolam, trimipramine, tripelennamine,

tyramine, verapamil, yohimbine

REFERENCE

Lam bert, W .E.; Meyer, E.; De L eenheer, A.P. System atic toxicological an alysis of basic d rugs by grad ient

elution of an alumina-based H PLC packing m aterial un der alkaline conditions. J.Anal.ToxicoL 1995,

19 7 3 - 7 8

SAMPLE

Matrix:

blood, tissue

Sample preparat ion: Whole blood, serum. 1 mL Whole blood or serum + 1 jxg cianopra-

mine + 1 mL water, vortex, add 1 mL 200 mM sodium c arbon ate, vortex, add 6 mL

hexane :

1-butanol

95:5, gently agitate for 30 min, centrifuge at 2500 g for 5 min. Remove

the organic layer and add it to 100 |xL 0.2% phosphoric acid, agitate gently for 30 min,

centrifuge for 5 min. Remove the organic layer and inject a 30 |JLL aliquot of th e aqueou s

layer. Tissue. 0.5 mL Liver homogenate + 10 |xg cianopramine + 500 |xL 2% sodium

tetraborate + 8 mL hexane :

1-butanol

95:5, gently agitate for 30 min, centrifuge at 2500

g for 5 min. Remove the organic layer and add it to 400 |xL 0.2% phosphoric acid, agitate

gently for 30 min, centrifuge for 5 min. Remove the organic layer and inject a 30 |xL

aliquot of the aqueous layer.

HPLCVARIABLES

Guard column: 15 X 3.2 7 |xm RP-18 Newguard (Applied Biosystems)

Column:

100 X 4.6 5 |xm Brownlee Spheri-5 RP-18

Mobile phase:

MeCN : 100 mM N aH

2

PO

4

: diethylamine 40:57 .5:2.5

Flow rate: 2


Detector: UV

220

CHROMATOGRAM


Internal s tandard: cianopramine (8.93)


50 ng/mL

OTHER SUBSTANCES

Simul taneous:

amitriptyline, amoxapine, benztropine, brompheniramine, chlorphenira-

mine, chlorpromazine, clomipramine, cyproheptadine, desipramine, dothiepin, doxepin,

fluoxetine, haloperidol, imipramine, loxapine, maprotiline, meperidine, mesoridazine,

me thadon e, m etoclopramide, m ianse rin, moclobemide, nomifensine, nordoxepin, norfluox-

etine, norpropoxyphene, northiaden, pentobarbital, pheniramine, promethazine, propox-

yphene, propranolol, protriptyline, quinidine, quinine, sulforidazine, thioridazine, thi-

othixene, tranylcypromine, trazodone, trihexyphenidyl, trim ipram ine, triprolidine

Noninterfering:

dextromethorphan, norphetidine, phenoxybenzamine, prochlorperazine,

trifluoperazine

Interfering:

diphenhydramine


27/36

KEYWORDS

serum; whole blood; liver

REFERENCE

M clntyre, I.M.; King, C.V.; Skafidis, S.; Drumm er, O.H. Du al ultraviolet wav elength high-performance

liquid chromatographic method for the forensic or clinical analysis of seventeen antidepressants and

some selected metabolites. J.Chromatogr 1993, 621 2 1 5 - 2 2 3

SAMPLE

Matrix: blood, tissue


Serum . 1 mL Seru m + 4 mL MeOH + 5 mL 2.5% perchloric acid,

shak e vigorously, centrifuge at 11000 g for 15 min. Add sup ern ata nt to 1 mL 4 M KOH,

centrifuge. Add supernatant (9 mL) to 10 mL diethyl ether:ethyl acetate 85:15, shake

for 15 min. Remove 8 mL of organic phase and evaporate it to dryness under a stream

of nitrogen. Dissolve residue in 200

JJLL

mobile ph ase buffer: MeO H 9 :1 , inject 100 |xL

aliquot. Tissue. 2 g Br ain tis sue + 10 mL 2.5% perchloric acid + 8 mL MeOH , homogenize,

centrifuge at 11000 g for 15 min. Add supernatant to 4 mL 4 M KOH, centrifuge. Add

supernatant to 20 mL diethyl ether-.ethyl acetate 3:1, shake for 15 min. Remove 8 mL

of organic phase and evaporate it to dryness under a stream of nitrogen. Dissolve residue

in 200 |xL mobile ph as e buffer:MeO H 9 : 1 , inject 100 |JLL aliquot.

HPLCVARIABLES

Column: 250 X 4.6 5 |jim Cosmosil 5C18

Mobile phase: M eOH : T H F : buffer 45 :1 7: 88 (Buffer w as 1% trieth ylam ine adjusted to pH

3.0 with phosphoric acid.)

Column tem peratu re: 40

Flow rate: 1


Detector:

UV 254

CHROMATOGRAM

Retent ion t ime:

14.2

Internal s tandard:

nortriptyline

Limit of detect ion: 10 ng/g (tissue)

OTHER SUBSTANCES

Extracted:

desipramine, imipramine

KEYWORDS

serum; rat; nortiptyline is IS

REFERENCE

Sugita, S.; Kobayashi, A.; Suzuki, S.; Yoshida, T.; Nakazawa, K. High-performance liquid chromato-

graphic determination of imipramine and its metabolites in rat brain. J.Chromatogr

1987,

421

412-417

SAMPLE

Matrix:

hair

Sample preparat ion: Wash hair in water, rinse 3 times with MeOH, dry, weigh. 5-25 mg

W ashed ha ir + 1 mL 1 M NaOH , hea t at 70° for 30 min, adjust pH to 9.5-10. 1 mL Ex tract

+ 1 |xg protriptyline + 1 mL wa ter + 1 mL 200 mM sodium ca rbona te buffer, mix, extract

with h exa ne: butan ol 95 :5 for 20 m in. Remove the organic layer a nd add it to 100 jxL

0.2% orthophosphoric acid, mix for 20 min, inject a 30 |xL aliquot of the aqueous layer.

HPLCVARIABLES

Guard column:

15 X 3.2 7 \xm Newguard RP-18


28/36

Column:

100 X 4.6 Spheri-5 RP-C18

Mobile phase: M eCN: buffer 40:60 (Buffer was 1.2 L 100 mM pH 7.0 NaH

2

PO

4

+ 30 mL

diethylamine.)

Flow rate: 2


Detector: UV 214

CHROMATOGRAM

Retention time: 5

Internal standard:

protriptyline (4)

OTHER SUBSTANCES

Extracted: amitriptyline, clomipramine, desipramine, dothiepin, doxepin, haloperidol,

imipramine, mianserin

KEYWORDS

may be interferences

REFERENCE

Couper, RJ.; Mclntyre, LM.; Drummer, O.H. Extraction of psychotropic drugs from human scalp hair.

J.Forensic ScL 1995, 40 8 3 - 8 6

SAMPLE

Matrix:

microsomal incubations

Sample preparation:

250 |JLL Microsomal incubation + 50 |xL 1 M HCl, cool on ice, add

desipramine, centrifuge at 16000 g for 5 min, inject an aliquot of the supernatant.

HPLCVARIABLES

Column:

300 X 3.9 jxBondapak C18

Mobile phase: MeCN: 50 mM pH 6 potassium phosphate buffer 35:65

Flow rate: 1.4

Detector: UV 220

CHROMATOGRAM


Internal standard: desipramine (9)

Limit of detection:

LOD 50 pmole

OTHER SUBSTANCES

Extracted: amitriptyline

Noninterfering:

ketoconazole, a-naphthoflavone, quinidine

KEYWORDS

human; liver

REFERENCE

Schmider, J.; Greenblatt, D.J.; von Moltke, L.L.; Harmatz, J.S.; Shader, R.L N-Demethylation of ami-

triptyline in vitro: Role of cytochrome P-450 3A (CYP3A) isoforms and effect of me tabolic in hib itors .

J.Pharm.Exp.Ther. 1995, 275 592-597

SAMPLE

Matrix: solutions

HPLCVARIABLES

Column: 250 X 4.6 5 jxm Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)


29/36

Mobile phase:

MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer

was 9 mL concentrated phosph oric acid and 10 mL triethy lam ine in 900 mL water, adjust

pH to 3.4 with dilute phosphoric acid, make up to 1 L.)

Flow rate: 0 .6


25

Detector: UV 229

CHROMATOGRAM

Retent ion t ime:

13.70 (A), 6.75 (B)

OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine,

albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atro-

pine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam,

bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine,

chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine,

chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimeti-

dine,

cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchi-

cine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal,

diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, do-

butamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine,

fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvox-

amine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hy-

dralazine, hydrochlorothiazide, hydrocodone, hydromorph one, hydroxychloroquine, hydro-

xyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac,

labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol,

mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol,

metformin, m ethadone, methdilazine, methocarbamol, methotrexate, metho trimeprazine,

methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol,

metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, na-

phazoline, naproxen, nifedipine, nizatidine, norepinephrine, oxazepam, oxycodone,

oxymetazoline, paroxetine, pem oline, pentazocine, pentob arbital, pentoxifylline, perph en-

azine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbuta-

zone, phenyltoloxamine, p henytoin, pimozide, pindolol, piroxicam, pramo xine, p razep am ,

prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine,

promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protrip-

tyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperi-

done, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfin-

pyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline,

thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide,

tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimepra-

zine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine,

zopiclone

KEYWORDS

some details of plasma extraction

REFERENCE

Koves, E.M. Use of high-perform ance liquid chrom atograph y-diode ar ra y detection in forensic toxicology.

J.Chromatogr.A 1995, 692 103-119

SAMPLE

Matrix:

solutions

HPLCVARIABLES

Guard co lumn:

30 X 2.1 Spheri-5 RP-8

Column:

220 X 2.1 Spheri-5 RP-8


30/36

Mobile phase: Gradient. A was 0.08% diethylamine and 0.09% phosphoric acid in water,

pH 2.3. B was MeCN: water 90:10 containing 0.08% diethylamine and 0.09% phosphoric

acid. A:B 95:5 for 2 min, to 0:100 over 15 min (?), maintain at 0:100 for 5 min.


Flow rate: 0.5

D etecto r: UV 200



OTHER SUBSTANCES

Sim ul tane ous : amitriptyline, desipramine, desmethyldoxepin, doxepin, imipramine

Also analyzed: amphetamine, chlordiazepoxide, chlorpromazine, desalkylflurazepam, di-

azepam, diethylpropion, ephedrine, fenfiuramine, flurazepam, mesoridazine, metham-

phetamine, norchlordiazepoxide, nordiazepam, oxazepam, phenterm ine, phenylpropano-

lamine, prazepam, promazine, thioridazine, thiothixene, trifluoperazine

REFERENCE

Rainin Catalog Ci-94 1994-5 Rainin Instrument Co. W oburn MA 1994, p. 7.24

SAMPLE

Matrix: solutions

Sa mple p re p ara ti on : Prepare a 1 mg/mL solution in MeOH, inject a 5 |xL aliquot.

HPLCVARIABLES

Colu mn: 250 X 4.6 5 |xm Lichrosphere cyanopropyl

M obile phase : Carbon dioxide: MeOH: isopropylamine 90:10:0.05


Flow rate: 3

Injection volume: 5

D etec tor: UV 220



OTHER SUBSTANCES

Simultaneous: amitriptyline, benactyzine, buclizine, desipramine, hydroxyzine, imipra-

mine, perphenazine, protriptyline, thioridazine

KEYWORDS

SFC;

pressure 200 bar

REFERENCE

Berger, T.A.; Wilson, W.H. Sepa ration of drugs by packed column supercritical fluid chrom atography. 2.

Antidepressants . J.Pharm.ScL 1994, 83 287-290

SAMPLE

Matrix: solutions

HPLCVARIABLES

Colu mn: 250 X 4.6 Zorbax RX

M obile phase : Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL trie-

thylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL trie-

thylamine 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.


31/36

Column temp erature: 30

Flow rate: 2

D etec tor: UV 210

OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline,

amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobar-

bital, a spirin, atenolol, atropin e, av ermec tin, bar bital, benzocaine, benzoic acid, benzotro-

pine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenor-

phine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal,

chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphe-

nesin, 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, dex-

tromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion,

diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxy-

late, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol,

ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide,

etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fen-

proporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluo-

xymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin,

glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide,

hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, im-

inostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproter-

enol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, Ii-

docaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine,

meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, me-

phentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesori-

dazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide,

methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldo-

pamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, meto-

prolol, mibolerone, m orphine, nadolol, nalorphine , naloxone, naltrexon e, naphazo line, na-

proxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid,

nitrazepam, norepinephrine, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone,

oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pento-

barbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendi-

metrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phen-

termine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam,

prednisolone, primidon e, probenecid, prog esterone, propiomazine, propranolol, propylpar-

aben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid,

quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol,

salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetam-

ide,

sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfa-

methoxizole, 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, tri-

amcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, tri-

methoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine,

warfarin, yohimbine, zoxazolamine

REFERENCE

Hill, D . W.; Kind, A. J. Reversed-pha se solvent grad ient HPLC retention indexes of drug s.

J.Anal.ToxicoL

1994,

18 233-242

SAMPLE

Matrix:

solutions


32/36

HPLCVARIABLES

Column:

150 X 4.6 5 ixm Adsorbosph ere C 18 (PEEK column) (retention tim es a re longer

and peaks broader with stainless steel column)

Mobi le phase:

MeC N: 20 mM p H 3.2 KH

2

PO

4

23.4:76.6 containing 0.05% nonylamine

F low rate: 1.2

Detector: UV

214

CHROMATOGRAM


OTHER SUBSTANCES

Simul taneous: amitriptyline, desmethyldoxepin, desipramine, doxepin, imipramine, lox-

apine, maprotiline, trazodone

REFERENCE

Alltech Chrom atography Catalog 300 Alltech Associates Inc. Deerfield IL 1993, p. 440

SAMPLE

Matrix: solutions


Inject an aliquot of a solution in mobile phase.

HPLCVARIABLES

Column:

250 X 4.6 Econosil C8

Mobile phase:

M eCN : buffer 3 0:7 0 (Buffer w as 20 mM K H

2

PO

4

and 14 mM triethylamine

adjusted to pH 3.0 with phosphoric acid.)


Detector: UV

210

CHROMATOGRAM

Retent ion t ime: 9 .9

Limit of quant i tat ion:

< 1 jig/mL

OTHER SUBSTANCES

Simul taneous: amitriptyline, amoxapine, carbamazepine, imipramine

Also analyzed:

desipramine, cyclobenzaprine, doxepin, maprotiline, protriptyline

KEYWORDS

UV spectra given

REFERENCE

Ryan, T.W. Identification and quantification of tricyclic antidepressants by UV-photodiode array detec-

tion with multicomponent analysis. J.Liq.Chromatogr. 1993, 16 1545-1560

SAMPLE

Matrix: solutions

Sample preparat ion: Dissolve in MeO H: w ate r 1:1 at a concen tration of 50 |xg/mL, inject

a 10 |xL aliquot.

HPLCVARIABLES

Column: 300 X 3.9 10 |xm jjuBondapak C18

Mobile phase: M eOH: acetic acid: triethy lam ine: water 60:1 .5:0.5 :38

F low rate: 1.5


Detector: UV


33/36

CHROMATOGRAM

Retent ion t ime: k 2.60

REFERENCE

Roos, R.W.; Lau-Cam, CA. General reversed-phase high-performance liquid chromatographic method

for the separation of drugs using triethylamine as a competing base. J.Chromatogr. 1986, 370

403-418

SAMPLE

Matrix: solutions


Prepare a 10 |xg/mL solution in MeOH, inject a 20 \xL aliquot.

HPLCVARIABLES

Column:

125 X 4.9 Spherisorb S5W silica

Mobile phase: MeOH containing 10 mM amm onium per chlorate and 1 mL/L 100 mM

NaOH in MeOH, pH 6.7

Flow rate: 2


Detector:

E, LeCarbone, V25 glassy carbon electrode, + 1.2 V

CHROMATOGRAM


OTHER SUB STANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albu-

terol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacy-

clonal, bamethane, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine,

benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol,

brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine,

butacaine, butethamate, chlorcyclizine, ehlorpheniramine, chlorphenoxamine, ehlorpren-

aline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clem astine,

clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine,

deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcar-

bamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, di-

methothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyrida-

mole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine,

ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine,

ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol,

fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol,

hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isox-

suprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamy-

lamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, mep-

tazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine,

methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine,

methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide,

metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, napha-

zoline, nicotine, nifedipine, nom ifensine, nortrip tyline, noscapine, orph enad rine, o xeladin,

oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, pen-

thienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phen-

butrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine,

phenm etrazine, phenom orphan, phenoperidine, phenothiazine, phenoxybenzamine, phen-

tolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pin-

dolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piri-

tramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine,

proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolin-

t ane , promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, pro-

thipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine,


34/36

quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine,

thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thiorida-

zine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcy-

promine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimetho-

benzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine,

verapamil, xylometazoline

REFERENCE

Ja ne , L; McKinnon, A.; Fla nag an,

R.

J. High-performance liquid ch roma tographic ana lysis of basic drug s

on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electro-

chemical oxidation detection. J.Chromatogr. 1985, 323 191-225

SAMPLE

Matrix:

urine


1 mL U rin e + 0.5 mL 1% trichloroacetic acid, centrifuge at 5200 g

for 10 min, filter (0.2 |xm), inject a 20 |xL aliquot of the nitrate.

HPLCVARIABLES

Column: 250 X 4 Lichrospher 5 |xm 60 RP-select B

Mobile phase:

Grad ient. MeC N: 50 mM pH 3.2 potassium pho sph ate buffer from 10:90 to

50:50 over 15 min.

F low rate: 1.5

Inject ion volume:

20

Detector: UV 190-370

CHROMATOGRAM

Retent ion t ime:

15

OTHER SUBSTANCES

Extracted:

benzoylecgonine, cocaine, diphenhydramine, ephedrine, lidocaine, morphine,

nordiazepam, norpropoxyphene, phenylpropanolamine

Also analyzed:

amitriptyline, amphetamine, codeine (different gradient), meperidine

REFERENCE

Li, S.; Gem perline, R J.; Briley, K.; Kazmierczak, S. Identification and qu anti tatio n of drug s of abuse in

urine using the generalized rank annihilation method of curve resolution. J.Chromatogr.B

1994,

655

213-223

SAMPLE

Matrix:

urine

Sample preparat ion: 500 jiL Ur ine + N-ethylno rdiazepam + chlorph eniram ine + 100 |JLL

buffer, centrifuge at 11000 g for 30 s, inject a 500 |xL aliqu ot on to column A w ith m obile

ph ase A, after 0.6 min back flush column A wit h mobile pha se A to was te for 1.6 m in,

elute column A with 250 JJIL mobile ph ase B, with 200 JJLL mobile pha se C , and with 1.15

mL mobile phase D. Elu te column A to wa ste un til drug s st ar t to emerge then elute onto

column B. Elute column B to waste until drugs start to emerge, then elute onto column

C. When all the drugs have emerged from column B, remove it from the circuit, elute

column C with mobile ph ase D, monitor th e effluent from column C. Flush column A with

7 mL mobile phase E, mobile phase D, and mobile phase A. Flush column B with 5 mL

mobile phase E then with mobile phase D. (Buffer was 6 M ammonium acetate adjusted

to pH 8.0 with 2 M KOH.)

HPLCVARIABLES

Column: A 10 X 2.1 12-20 (xm PR P- I sph erical poly(styrene-d ivinylbenzene) (Ham ilton); B

10 X 3.2 11 |xm Am inex A-28 (Bio-Rad); C 25 X 3.2 5 jxm C8 (Phenom enex) + 150 X 4.6

5 |xm silica (Macherey-Nagel)


35/36

Mobile phase:

A 0.1% pH 8.0 po tassiu m bora te buffer; B 6 mM KH

2

PO

4

containing 5 mM

tetramethylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50

w ith phosp horic acid; C M eCN : buffer 40 :60 (Buffer wa s 6 mM K H

2

PO

4

containing 5 mM

tetramethylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50

w ith pho sphoric acid.); D MeC N: buffer 33 :67 (Buffer w as 6 mM KH

2

PO

4

containing 5

mM tetramethylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to

6.50 with phosph oric acid.); E M eC N: buffer 70 :30 (Buffer w as 6 mM KH

2

PO

4

containing

5 mM tetramethylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to

6.50 with phosphoric acid.)

Column temperature: 40 (B, C only)

F low rate: A 5; B-E 1

Inject ion volume:

500

Detector: UV 210; UV 235

CHROMATOGRAM

Retention time: k 3.3

Internal s tandard:

N-ethylnordiazepam (k' 2.1), chlorpheniramine (k' 5.9)


300 ng/mL

OTHER SUBSTANCES

Extracted:

amphetamine, benzoylecgonine, caffeine, cotinine, diazepam, ephedrine, lido-

caine, nordiazepam, oxazepam, pentazocine, phen me trazine, phenobarbital, ph enterm ine,

phenylpropanolamine, secobarbital, amitriptyline, codeine, flurazepam, hydrocodone, hy-

dromorphone, imipramine, methadone, morphine

Interfering:

desipramine, diphenhydramine, metham phetamine

KEYWORDS

column- switchin g

REFEREN

Documents

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