Midazolam 1

8/20/2019 Midazolam 1

http://slidepdf.com/reader/full/midazolam-1 1/6

SAMPLE

Matrix: aqueous humor, blood, tissue, urine

Sample preparat ion:

Hom ogenize ti ss ue 1:2 (w/v). 1 mL Sam ple + 100 |xL 10 img/mL

m ethaq ualon e + 1 mL amm onium chloride/amm onium hy droxide buffer (pH 9.2) + 3 mL

n-butyl chloride, mix, centrifuge. Remove the organic layer and evaporate it to dryness

under nitrogen at 45°, reconstitute the residue with 50 |xL mobile phase, inject a 20 |xL

aliquot.

HPLCVARIABLES

Column: 100 X 8 10 |xm ixBondapak C18

Mobile phase:

M eC N: buffer 40 :60 , pH 3.3 (Buffer w as 150 mL 100 mM K H

2

PO

4

made up

to 1 L, pH adjusted to 3.3 with 100 mM phosphoric acid.)

Flow ra te:

2.5

Inject ion volume:

20

Detector: UV

220

CHROMATOGRAM

Retent ion t ime: 3.93

Internal s tandard:

methaqualone (6.42)

KEYWORDS

plasma; liver; kidney

REFERENCE

Ferslew, K.E.; Hagardorn, A.N.; McCormick, W.F. Postmortem determination of the biological distri-

bution of sufentanil and midazolam after an acute intoxication. J.Forensic Sci., 1989, 34, 249—257

SAMPLE

Matrix:

blood


Condition a Sep-Pak C18 SPE cartridge with water, MeOH, and 100

mM ammonium acetate. 5 mL Plasma + 250 ng detomidine, add to the SPE cartridge,

wash with 100 mM am mon ium acetate, elute with MeO H: 100 mM am monium acetate

75:25. Evaporate the eluate to dryness under reduced pressure, reconstitute the residue

in 200 |xL mobile phase, inject a 50 |xL aliquot.

HPLCVARIABLES

Column:

150 X 4.6 5 |xm Hitachi gel

Mobile phase: 3056

Mobile phase:

MeO H: 100 mM am monium acetate 6 5:35

Flow rate: 1

Inject ion volume: 50

Detector:

M S, H itach i M-1000, APC I interface, drift voltage 21 V, neb ulizer 260°, vapo rizer

399°, multiplier voltage 1500 VF, m/z 326

Midazolam

Molecular formula: C

18

H

13

CIFN

3

Molecular weight: 325.8

CAS Registry No : 59467-70-8 midazolam), 59467-96-8 midazolam

hydrochloride), 59467-94-6 midazolam maleate)



CHROMATOGRAM

Retent ion t ime:

10.5

Internal s tandard: detomidine (m/z 187) (6.5)

Limit of quant i tat ion:

1-2 ng/mL

OTHER SUBSTANCES

Extracted: atipamazole, medetomidine

KEYWORDS

pig; plasma; pharmacok inetics; S PE

REFERENCE

Kanazawa, H.; Nishimura, R.; Sasaki, N.; Takeuchi, A.; Takai, N.; Nagata, Y.; Matsushima, Y. Deter-

mination of medetomidine, atipamazole and midazolam by liquid chromatography-mass spectrome-

try. Biomed.Chromatogr., 1995, 9, 188-191

SAMPLE

Matrix:

blood


1 mL P las m a + 300 jxL 100 mM p H 9 bo rate buffer + 25 |xL flur-

azepam in EtOH + 5 mL diethyl ether, mix at 60 rpm for 10 min, centrifuge at 15° at

1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream

of nitrogen at 45°, reconstitute the residue in 100 |xL mobile phase, inject an aliquot.

HPLCVARIABLES

Column:

150 X 4.6 5 |xm Spherisorb CN

Mobile phase: M eOH : isopropanol 75:2 5 contain ing 0.015 perchloric acid

F lo w rate : 1.5

Detector: UV 215

CHROMATOGRAM

Retent ion t ime: 4 .7

Internal s tandard:

flurazepam (6.2)

Limit of quant i tat ion: 2 ng/mL

OTHER SUBSTANCES

Extracted: metabolites

KEYWORDS

plasma

REFERENCE

Lehmann, B.; Boulieu, R. Determination of midazolam and its unconjugated 1-hydroxy metabolite in

human plasma by high-performance liquid chromatography. J.Chromatogr.B, 1995, 674, 138-142

SAMPLE

Matrix:

blood

Sample preparat ion: 600 JJLL Pl asm a + 600 x̂L IS solution, s ha ke, centrifuge at 1500 g

for 3 min, inject a 400 |xL aliquot onto column A with mobile phase A, elute with mobile

ph ase A for 4 min, backflush column A with mobile pha se A for 1.5 m in, backflush column

A with mobile ph ase B for 4.5 m in, backflush conten ts of column A onto column B w ith

mobile phas e C and st ar t th e grad ient. After 3 min remove column A from circuit, monitor

effluent from column B. (IS solution was 2.5 mL 200 |xg/mL flurazepam in MeCN + 3.6

mL 2 M NaOH, add 200 mL MeCN, mak e up to 1 L with water.)



HPLC VARIABLES

C o l u m n : A 17 X 4.6 37-50 jxm Bondapak C18 C orasil; B 4 X 4 5 |xm LiChrospher 60 RP-

select B + 250 X 4 5 |xm LiChrospher 60 RP-select B

M o b i l e p h a s e : A 100 mM NaOH; B 2.7 g/L K H

2

PO

4

adjusted to pH 8.0 with 2 M NaOH;

C Gradient. I was 2.7 g/L KH

2

PO

4

adjusted to pH 2.4 with 85 phosphoric acid. II was

MeCN. I:II from 76:24 to 66:34 over 11 min.

F l o w r a t e :

A 1; B 1; C 1.5

I n j e c t i o n v o l u m e : 400

D e t e c t o r : UV 230

CHROMATOGRAM

Retention time: 20.5

Internal standard: flurazepam (21.5)

Limit of detection: 2 ng/mL

Limit of quantitation: 10 ng/mL

OTHER SUBSTANCES


KEYWORDS

plasma

REFERENCE

Lauber, R.; M osiman, M.; Biihrer, M.; Zbinden, A.M. Automated determination of midazolam in human

plasma

by

high-performance liquid chrom atograph y us ing column switching. J.Chromatogr.B,

1994,

654,

6 9 - 7 5

SAMPLE

Matrix: blood

Sam ple p re p a r a t io n : Cond ition a 12 mL 500 mg PrepSep Cl SPE cartridge with 3 mL

MeOH and 3 mL water. 1 mL Plasma + 100

\xh

3 |xg/mL midazolam in MeOH, mix, add

to SPE cartridge, w ash with two 3 mL portions of water, wash with two 1 mL portions

of MeOH: water 30:70, elute with two 1 mL portions of MeOH: 50 mM pH 9.0 (NHJ

2

HPO

4

90:10, evaporate the eluents under vacuum, dissolve the residue in 200 JJLL mobile p has e,

inject a 100 |xL aliquot.

HPLCVARIABLES

C o l u m n : 100 X 4.6 5 |xm Spherisorb C8

M obi le p ha se : MeCN: MeOH: 20 mM (NH

4

)H

2

PO

4

5:35:60

containing 2 mL/L 200 mM te-

trabutylammonium bromide, final pH adjusted to 4.10

C o lu m n t e m p e r a t u r e : 30

F l o w r a t e : 1.5

I n j e c t i o n v o l u m e : 100

D e t e c t o r : UV 254

CHROMATOGRAM


Internal standard: clonazepam (12.4)

Limit of quantitation: 15 ng/mL

OTHER SUBSTANCES


KEYWORDS

plasma;

SPE



REFERENCE

Mastey, V.; Pan neto n, A .-C; D onati, R; Varin, F. De term ination of midazolam and two of its m etabolites

in human plasma by high-performance liquid chromatography. J.Chromatogr.B, 1994, 655, 305-310

SAMPLE

Matrix:

blood

Sample preparat ion: Autom ated S PE by ASPEC system. Condition a C18 Clean-Up SPE

cartridge (CEC

18111,

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

Plas m a + 1 mL 400 ng/mL pro triptyline in water, vortex, add to column, wa sh with 3

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

HPLCVARIABLES

Guard column: LC-8-DB (Supelco)

Column:

150 X 4.6 LC-8-DB (Supelco)

Mobile phase: MeCN.buffer 35:65 (Buffer was 10 mL/L triethylamine in water adjusted

to pH 5.5 with glacial acetic acid.)

Flow rate: 2

Inject ion vo lum e:

100

Detector: UV

228

CHROMATOGRAM

Retention tim e: 7 6

Internal s tandard:

protriptyline (4)

OTHER SUBSTANCES

Extracted:

acetazolamide, amitriptyline, chlordiazepoxide, chlorimipramine, chlorproma-

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

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

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

imipramine, nordiazepam, nordoxepin, norfluoxetine, nortriptyline, norverapamil, pen-

tazocine, promazine, propafenone, propoxyphene, propranolol, protriptyline, quinidine,

temazepam, trazodone, trimipramine, verapamil

Noninterfering:

acetaminophen, acetylmorphine, amiodarone, amob arbital, amphetam 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

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

Sample preparation:

Condition a 100 mg Bond-Elut C2 SPE cartridge with 1 volume

MeOH and 1 volume 10 mM pH 8.0 phosphate buffer. 1 mL Plasma + 5 |xg prazepam +

100 |JLL 1 M pH 8.0 potassium phosphate buffer, mix, add to the SPE cartridge, wash with

3 volumes of water, wash w ith 1 mL MeOH: water 30:70, wash with 1 mL water, elute

with 1 mL MeOH-.water 70:30, elute with 1 mL water. Evaporate the eluate to dryness,

reconstitute with 200 |xL mobile phase, inject an aliquot.

HPLC VARIABLES

Column: 35 X 4.6 5 jxm Ultrabase C18

Mobile phase:

MeOH: water 60:40

Flow rate: 1

Injection volume:

20

Detector: UV 217

CHROMATOGRAM

Retention time: 4

Internal standard:

prazepam (8)

Limit of detection: 3 ng/mL

Limit of quantitation:

5 ng/mL

KEYWORDS

plasma; SPE

REFERENCE

Berrueta, L.A.; Gallo, B.; Vincente, F. Rapid determination of midazolam in plasma using SPE and

HPLC. Am .Lab., 1993, 25 Dec), 20R-2OT

SAMPLE

Matrix: blood

Sample preparation:

Condition a Sep-Pak C18 SPE cartridge with water, MeOH, and 100

mM ammonium acetate. Add 200 |xL plasma to the SPE cartridge, wash with 100 mM

ammonium acetate, elute with MeOHrIOO mM ammonium acetate 3:1. Evaporate the

eluate to dryness under reduced pressure, dissolve the residue in 200

|JIL

mobile phase,

inject a 20 JULL aliquot.

HPLCVARIABLES

Column: 150 X 4.6 Hitachi gel 3056 octadecylsilica

Mobile phase:

MeOH: 100 mM ammonium acetate 60:40

Flow rate: 1

Injection volume:

20

Detector: MS, Hitachi M1000, APCI, nebulizer 260°, vaporizer 399°

CHROMATOGRAM


Limit of detection:

0.5-2.5 ng/mL

OTHER SUBSTANCES

Simultaneous: atipamezole, atropine, butorphanol, flumazenil, ketamine, medetomidine,

xylazine

KEYWORDS

plasma; SPE; dog



REFERENCE

Kanazawa,

H.;

Nag ata, Y.; Matsushim a,

Y;

Takai,

N.;

Uchiyama,

H.;

Nishimura,

R.;

T akeuchi,

A.

Liquid

chromatography-mass spectrometry

for the

d etermination

of

m edetomidine

and

other anaesthetics

in plasma. J.Chromatogr., 1993

631

215-220

SAMPLE

Matrix:

blood

Sample preparat ion: Condition

a 100 mg C18

Bond-Elut

SPE

cartridg e with

2 mL

MeOH

and 2 mL water. 1 mL Plasma + 100 jxL 10 jxg/mL elimazolam in MeOH + 1 mL

MeC N: water 30:70 , vortex

for 10 s,

centrifuge

for 5 min at

4000

g, add to the SPE

cartridge, wash with 2 mL MeCN: water 15:85, let dry for 3-4 min, elute with four 200

jxL aliquots of MeOH. Evaporate the eluate un der nitrogen, tak e up the residue in 100

IxL MeOH, inject

a 20 |xL

aliquot.

HPLCVARIABLES

Guard co lumn:

4 X 4 5 |xm

LiChrosorb

100 RP 18

Column:

125 X 4 5 |xm LiChrospher 100 RP 18 endcapped

Mobile phase: MeC N:MeOH :THF:buffer 2 8:2 5:2:5 0 (Prepare a 1 M pH 5.6 phosphate

buffer from

94.8 mL 1 M

KH

2

PO

4

+ 5.2 mL 1 M

K

2

HPO

4

. Dilute

10 mL of

th is buffer

to

1 L to give the 10 mM pH 5.6 phosphate buffer used in the mobile phase.)

Flow ra te: 1.3

Inject ion vo lum e:

20

Detector:

UV 254

CHROMATOGRAM

Retent ion t ime:

7.48

Internal s tandard:

elimazolam (9.79)

Limit of q uant i ta t ion:

50 ng/mL

OTHER SUBSTANCES


KEYWORDS

plasma; SPE

REFERENCE

Sautou, V.; C hopineau, J.; Terrisse, M .P.; Bastide , P. Solid-phase extraction of midazolam and two of its

metabolites from plasma

for

high-performance liquid chromatographic analysis. J.Chrom atogr.,

1991

571, 298-304

SAMPLE

Matrix: blood


1 mL

Plasma

+ 25

JJLL EtOH

+ 25

|JLL

3

|xg/mL

ISl and 7.6

jxg/mL

IS 2 in EtOH + I mL 100 mM Na

2

HPO

4

adjusted to pH 10.5 with NaOH + 5 mL diethyl

ether : dichloromethane 60:40 , vortex for 30 s, centrifuge at 4° at 2000 g for 10 min. Re-

move

the

organic phase

and add it to 1 mL 100 mM

Na

2

HPO

4

adjusted

to pH 10.5

with

NaOH, vortex for 30 s, centrifuge at 4° at 2000 g for 5 min. Remove the organic layer and

evaporate

it to

dryness un der

a

stream

of

nitrogen

at 40°,

reconstitute

the

residue

in 50

JULL

mobile phase, inject a 1-15

JJLL

aliquot.

HPLCVARIABLES

Column:

100 X 4.6 3 fxm

CP-M icrospher

C18

(Chrompack)

Mobile phase:

Gradient. A was M eOH:buffer 1:2. B was MeOH : water 80:20. A:B 93.8:

6.2 for 5.5 min, to 60:40 over 0.15 min, m ainta in at 60:40 for 11.3 min, to 2.5:97.5 over

0.5 min, maintain at 2.5:97.5 for 3.5 min, return to initial conditions over 0.5 min (Buffer

2 4

Documents

Midazolam 1