sample preparation assignment

8/12/2019 Sample Preparation Assignment

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I. BLOOD SAMPLE1. SAMPLE

Matrix: blood

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

volumes water. Add 1 mL serum then 200 L 700 ng/mL promazine in MeOHrO. 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 (JLL 10 mM ammonium acetate in MeOH, wait for 30 s,

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

them to dryness at 56-8 under compressed air. Reconstitute with 200 L mobile

phase, vortex 10 s, inject 75-100 L aliquot. (MeOH/water was 500 mL MeOH:water 65:

35 plus 25 L concentrated HCl.)

HPLCVARIABLES

Column: 250 X 4.6 5 jim Supelco silica

Mobile phase: EtOH:MeCN:t-butylamine 98:2:0.05 (Mix 1 gallon EtOH with 77 mL MeCN

and 1.9 mL t-butylamine.)

Flow rate: 2

Injection volume: 75-100

Detector: UV 254

CHROMATOGRAM

Retention time: 3.6, 3.3 (cis isomer)

Internal standard: promazine (5.2)

Limit of detection: 2 ng/mL

OTHER SUBSTANCES

Extracted: amitriptyline, desipramine, desmethyldoxepin, imipramine, nortriptyline,

protriptyline

Simultaneous: N-acetylprocainamide, procainamide, zimeldine, morphine, codeine,

trifluoperazine, 2-hydroxydesipramine, desmethylchlordiazepoxide, buprion, diazepam,

demoxepam, chlordiazepoxide, propoxyphene, dextropropoxyphene, cocaine, oxapam,

trimipramine, mianserin, trimeprazine, loxepin, fluphenazine, methadone, trifluopromazine,

phenteramine, chlorimipramine

Noninterfering: thiopropazineInterfering: thioridazine, hydroxyamoxapine, meperidine, chlorpromazine, disopyramide,


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amphetamine, 2-hydroxyimipramine, iprindole, pyrilamine, promethazine, prolixin,

amoxapine

KEYWORDS

serum; normal phase

REFERENCE

Beierle,F.A.; Hubbard,R-W. Liquid chromatographic separation of antidepressant drugs: I.

Tricyclics, Ther.DrugMonit, 1983, 5, 279-292.

2. SAMPLEMatrix: 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 L 200

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

HPLC VARIABLES

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

Mobile phase: MeCN:500 mM NaH2PO4 35:65 adjusted to pH 2.2 with phosphoric acidFlow rate: 0.8

Injection volume: 20

Detector: UV 210

CHROMATOGRAM

Retention time: 3.3


OTHER SUBSTANCES

Simultaneous: nortriptyline, amitriptyline

KEY WORDS

serum

REFERENCE


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Van Damme,M.; Molle,L.; Abi Khalil,F. Useful sample handlings for reversed phase high

performance liquid chromatography in emergency toxicology, J.Toxicol.Clin.ToxicoL, 1985,

23, 589-614.

3. SAMPLEMatrix: blood

Sample preparation: Condition a 1 mL Analytichem cyanopropyl SPE cartridge with 1

mL water and 1 mL MeOH, do not allow to dry. Add 1 mL serum + 250 (xL 0.2 g/mL

imipramine in 50 mM sodium n-heptanesulfonic acid to the SPE cartridge, wash with 1

mL water, 1 mL MeOH:water 50:50, air dry cartridge, elute with 1 mL MeOH:triethylamine

99.2:0.8, evaporate eluate to dryness under a stream of nitrogen at 40, reconstitute

residue with 250 L mobile phase, inject a 50 L aliquot.

HPLC VARIABLES

Column: 150 X 4.6 5(xm Spherisorb cyanopropyl

Mobile phase: MeOH:20 mM phosphoric acid containing 0.05% N,N-diethyloctylamine 55:

45, pH was 2.4

Flow rate: 1.5

Injection volume: 50Detector: UV 214

CHROMATOGRAM

Retention time: 6

Internal standard: imipramine (9)


OTHER SUBSTANCES

Simultaneous: metabolites

KEYWORDS

serum; SPE

REFERENCE


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Emm,T.; Lesko,L.J.; Perkal,M.B. Simultaneous determination of doxepin and nordoxepin in

serum using high-performance liquid chromatography,J.Chromatogr., 1987,419, 445-451.

II. URINE BLOOD SAMPLE1. SAMPLE

Matrix: blood, urine

Sample preparation: Add 500 L 3 M ammonia solution and 7 mL n-pentane:isopropanol

95:5 to 2 mL plasma containing 200 ng/mL nortriptyline or 2 mL urine containing 400 ng/mL

nortriptyline, shake in an overhead shaker for 20 min, let stand for 10 min. Transfer the

upper organic layer to a tube containing 1 mL 100 mM HCl, shake for 20 min, let stand for 5

min. Aspirate the organic phase to waste, wash the remaining aqueous layer with 3 mL

pentane by shaking for 10 min. Add 500 L3 M ammonia solution and 6 mL a-

pentane:isopropanol 95:5 to the washed aqueous layer, shake for 20 min, evaporate the

organic layer under a stream of nitrogen at 65, reconstitute the residue with 160 L mobile

phase, inject a 60 L aliquot.

HPLC VARIABLES

Column: 150 X 4.5 3 m Spherisorb silica

Mobile phase: MeOH:hexane:nonylamine 5:95:0.3

Flow rate: 1


Detector: UV 254

CHROMATOGRAM

Retention time: 4.0 (cis), 4.5 (trans)

Internal standard: nortriptyline (10)

OTHER SUBSTANCES

Extracted: metabolites, N-desmethyldoxepin

KEYWORDS

dog; human; plasma; normal phase

REFERENCE


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Yan,J.; Hubbard,J.W.; McKay,G.; Midha,K.K. Stereoselective and simultaneous measurement

of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-

performance liquid chromatography,J.Chromatogr.B, 1997, 691, 131-138.

III.HAIR SAMPLE1. SAMPLE

Matrix: hair

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

Washed hair + 1 mL 1 M NaOH, heat at 70 for 30 min, adjust pH to 9.5-10. 1 mL Extract + 1

|xg protriptyline + 1 mL water + 1 mL 200 mM sodium carbonate buffer, mix, extract with

hexane:butanol 95:5 for 20 min. Remove the organic layer and add it to 100 JULL 0.2%

orthophosphoric acid, mix for 20 min, inject a 30 JULL aliquot of the aqueous layer.

HPLC VARIABLES

Guard column: 15 X 3.2 7 u-m Newguard RP-18

Column: 100 X 4.6 Spheri-5 RP-C18

Mobile phase: MeCN:buffer 40:60 (Buffer was 1.2 L 100 mM pH 7.0 NaH2PO4 + 30 Ml

diethylamine.)

Flow rate: 2


Detector: UV 214

CHROMATOGRAM

Internal standard: protriptyline (4)

OTHER SUBSTANCES

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

mianserin, nortriptyline

KEYWORDS

may be interferences

IV.SOLUTION SAMPLE1.

SAMPLEMatrix: solutions


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HPLC VARIABLES

Guard column: 30 X 2.1 Spheri-5 RP-8

Column: 220 X 2.1 Spheri-5 RP-8

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.

Column temperature: 50

Flow rate: 0.5

Detector: UV 200

CHROMATOGRAM

Retention time: 13

OTHER SUBSTANCES

Simultaneous: desmethyldoxepin, desipramine, nortriptyline, imipramine, amitriptyline

Also analyzed: amphetamine, chlordiazepoxide, chlorpromazine, desalkylflurazepam,

diazepam, diethylpropion, ephedrine, fenfluramine, flurazepam, mesoridazine,

methamphetamine, norchlordiazepoxide, nordiazepam, oxazepam, phentermine,phenylpropanolamine, prazepam, promazine, thioridazine, thiothixene, trifluoperazine

REFERENCE

Rainin Catalog, Cl-94, 1994, p. 7.24.

sample preparation assignment

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