Midazolam

Molecular formula: C18H13CIFN3

Molecular weight: 325.8
CAS Registry No.: 59467-70-8 (midazolam), 59467-96-8 (midazolam
hydrochloride), 59467-94-6 (midazolam maleate)

SAMPLE
Matrix: aqueous humor, blood, tissue, urine
Sample preparation: Homogenize tissue 1:2 (w/v). 1 mL Sample + 100 |xL 10 img/mL
methaqualone + 1 mL ammonium chloride/ammonium hydroxide 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: MeCN: buffer 40:60, pH 3.3 (Buffer was 150 mL 100 mM KH2PO4 made up
to 1 L, pH adjusted to 3.3 with 100 mM phosphoric acid.)
Flow rate: 2.5
Injection volume: 20
Detector: UV 220
CHROMATOGRAM
Retention time: 3.93
Internal standard: methaqualone (6.42)
KEYWORDS
plasma; liver; kidney
REFERENCE
Ferslew, K.E.; Hagardorn, A.N.; McCormick, W.F. Postmortem determination of the biological distribution of sufentanil and midazolam after an acute intoxication. J.Forensic Sci., 1989, 34, 249257

SAMPLE
Matrix: blood
Sample preparation: 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 ammonium acetate, elute with MeOH: 100 mM ammonium 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: MeOH: 100 mM ammonium acetate 65:35
Flow rate: 1
Injection volume: 50
Detector: MS, Hitachi M-1000, APCI interface, drift voltage 21 V, nebulizer 260, vaporizer
399, multiplier voltage 1500 VF, m/z 326

CHROMATOGRAM
Retention time: 10.5
Internal standard: detomidine (m/z 187) (6.5)
Limit of quantitation: 1-2 ng/mL
OTHER SUBSTANCES
Extracted: atipamazole, medetomidine
KEYWORDS
pig; plasma; pharmacokinetics; SPE
REFERENCE
Kanazawa, H.; Nishimura, R.; Sasaki, N.; Takeuchi, A.; Takai, N.; Nagata, Y.; Matsushima, Y. Determination of medetomidine, atipamazole and midazolam by liquid chromatography-mass spectrometry. Biomed.Chromatogr., 1995, 9, 188-191

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 300 jxL 100 mM pH 9 borate buffer + 25 |xL flurazepam 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: MeOH: isopropanol 75:25 containing 0.015% perchloric acid
Flow rate: 1.5
Detector: UV 215
CHROMATOGRAM
Retention time: 4.7
Internal standard: flurazepam (6.2)
Limit of quantitation: 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 preparation: 600 JJLL Plasma + 600 ^xL IS solution, shake, centrifuge at 1500 g
for 3 min, inject a 400 |xL aliquot onto column A with mobile phase A, elute with mobile
phase A for 4 min, backflush column A with mobile phase A for 1.5 min, backflush column
A with mobile phase B for 4.5 min, backflush contents of column A onto column B with
mobile phase C and start the gradient. 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, make up to 1 L with water.)

HPLC VARIABLES
Column: A 17 X 4.6 37-50 jxm Bondapak C18 Corasil; B 4 X 4 5 |xm LiChrospher 60 RPselect B + 250 X 4 5 |xm LiChrospher 60 RP-select B
Mobile phase: A 100 mM NaOH; B 2.7 g/L KH2PO4 adjusted to pH 8.0 with 2 M NaOH;
C Gradient. I was 2.7 g/L KH2PO4 adjusted to pH 2.4 with 85% phosphoric acid. II was
MeCN. I:II from 76:24 to 66:34 over 11 min.
Flow rate: A 1; B 1; C 1.5
Injection volume: 400
Detector: 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
Extracted: metabolites
KEYWORDS
plasma
REFERENCE
Lauber, R.; Mosiman, M.; Biihrer, M.; Zbinden, A.M. Automated determination of midazolam in human
plasma by high-performance liquid chromatography using column switching. J.Chromatogr.B, 1994,
654, 69-75

SAMPLE
Matrix: blood
Sample preparation: Condition 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, wash 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 (NHJ2HPO4
90:10, evaporate the eluents under vacuum, dissolve the residue in 200 JJLL mobile phase,
inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 5 |xm Spherisorb C8
Mobile phase: MeCN: MeOH: 20 mM (NH4)H2PO4 5:35:60 containing 2 mL/L 200 mM tetrabutylammonium bromide, final pH adjusted to 4.10
Column temperature: 30
Flow rate: 1.5
Injection volume: 100
Detector: UV 254
CHROMATOGRAM
Retention time: 10.2
Internal standard: clonazepam (12.4)
Limit of quantitation: 15 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEYWORDS
plasma; SPE

REFERENCE
Mastey, V.; Panneton, A.-C; Donati, R; Varin, F. Determination of midazolam and two of its metabolites
in human plasma by high-performance liquid chromatography. J.Chromatogr.B, 1994, 655, 305-310

SAMPLE
Matrix: blood
Sample preparation: 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
Plasma + 1 mL 400 ng/mL protriptyline in water, vortex, add to column, wash 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
Injection volume: 100
Detector: UV 228
CHROMATOGRAM
Retention time: 7.6
Internal standard: protriptyline (4)
OTHER SUBSTANCES
Extracted: acetazolamide, amitriptyline, chlordiazepoxide, chlorimipramine, chlorpromazine, desipramine, dextromethorphan, diazepam, diphenhydramine, doxepin, encainide,
fentanyl, flecainide, fluoxetine, flurazepam, haloperidol, hydroxyethylflurazepam, ibuprofen, imipramine, lidocaine, maprotiline, methadone, methaqualone, mexiletine, norchlorimipramine, nordiazepam, nordoxepin, norfluoxetine, nortriptyline, norverapamil, pentazocine, promazine, propafenone, propoxyphene, propranolol, protriptyline, quinidine,
temazepam, trazodone, trimipramine, verapamil
Noninterfering: acetaminophen, acetylmorphine, amiodarone, amobarbital, amphetamine,
bendroflumethiazide, benzocaine, benzoylecgonine, benzthiazide, butalbital, carbamazepine, chlorothiazide, clonazepam, cocaine, codeine, cotinine, cyclosporine, cyclothiazide,
desalkylflurazepam, diamorphine, dicumerol, ephedrine, ethacrynic acid, ethanol, ethchlorvynol, ethosuximide, furosemide, glutethimide, hydrochlorothiazide, hydrocodone,
hydroflumethiazide, hydromorphone, lorazepam, mephentermine, meprobamate, methamphetamine, metharbital, methoxsalen, methoxyphenteramine, methsuximide, methylcyclothiazide, metoprolol, MHPG, monoacetylmorphine, morphine, normethsuximide,
oxazepam, oxycodone, oxymorphone, pentobarbital, phencyclidine, phenteramine, phenylephrine, phenytoin, polythiazide, primidone, prochlorperazine, salicylic acid, sulfanilamide, THC-COOH, theophylline, thiazolam, thiopental, thioridazine, tocainide, trichloromethiazide, 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 with 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

Retention time: 12.7

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.; Nagata, Y.; Matsushima, Y; Takai, N.; Uchiyama, H.; Nishimura, R.; Takeuchi, A. Liquid
chromatography-mass spectrometry for the determination of medetomidine and other anaesthetics
in plasma. J.Chromatogr., 1993, 631, 215-220

SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg C18 Bond-Elut SPE cartridge with 2 mL MeOH
and 2 mL water. 1 mL Plasma + 100 jxL 10 jxg/mL elimazolam in MeOH + 1 mL
MeCN: 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 under nitrogen, take up the residue in 100
IxL MeOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 4 X 4 5 |xm LiChrosorb 100 RP 18
Column: 125 X 4 5 |xm LiChrospher 100 RP 18 endcapped
Mobile phase: MeCN:MeOH:THF:buffer 28:25:2:50 (Prepare a 1 M pH 5.6 phosphate
buffer from 94.8 mL 1 M KH2PO4 + 5.2 mL 1 M K2HPO4. Dilute 10 mL of this buffer to
1 L to give the 10 mM pH 5.6 phosphate buffer used in the mobile phase.)
Flow rate: 1.3
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 7.48
Internal standard: elimazolam (9.79)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEYWORDS
plasma; SPE
REFERENCE
Sautou, V.; Chopineau, 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.Chromatogr., 1991,
571, 298-304

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 25 JJLL EtOH + 25 |JLL 3 |xg/mL ISl and 7.6 jxg/mL
IS2 in EtOH + I m L 100 mM Na2HPO4 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. Remove the organic phase and add it to 1 mL 100 mM Na2HPO4 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 under 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-Microspher C18 (Chrompack)
Mobile phase: Gradient. A was MeOH: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, maintain 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
was 6 g/L NaH2PO4 and 1 mL/L triethylamine adjusted to pH 7.00 with NaOH.)