Gemfibrozil

Molecular formula: C15H22O3 Molecular weight: 250.3 CAS Registry No.: 25812-30-0

SAMPLE Matrix: blood Sample preparation: Adjust pH of plasma to 4-5 with phosphoric acid (about 30 |JLL phosphoric acid per 4 mL of plasma). 500 fxL Acidified plasma + 100 jxL 2 |xg/mL flurbiprofen in MeCN: water 30:70 + 1.3 mL MeCN, vortex, centrifuge at 1000 g for 15 min. Remove the supernatant and add it to 800 JJLL 1 M pH 3.0 glycine buffer and 4 mL ethyl acetate, shake horizontally at 70 rpm for 15 min, centrifuge at 1000 g for 15 min. Remove the organic layer and evaporate it to dryness in an evacuated centrifuge, reconstitute the residue in 250 JULL mobile phase, inject a 10-100 JULL aliquot. HPLC VARIABLES Column: 250 X 4.6 5 |xm Ultrasphere cyano Mobile phase: MeCN: 10 mM tetrabutylammonium sulfate 28:72, final apparent pH adjusted to 3.5 Flow rate: 1 Injection volume: 10-100 Detector: F ex 284 em 316 CHROMATOGRAM Retention time: 20.9 Internal standard: flurbiprofen (17.6) Limit of detection: 100 ng/mL OTHER SUBSTANCES Extracted: metabolites, glucuronide KEYWORDS plasma REFERENCE
Sallustio, B.C.; Fairchild, B.A. Biosynthesis, characterization and direct high-performance liquid chromatographic analysis of gemfibrozil l-O-3-acylglucuronide. J.Chromatogr.B, 1995, 665, 345-353

SAMPLE Matrix: blood Sample preparation: 0.5 mL Plasma H 0.5 mL 10 |xg/mL IS in MeCN :pH 7.4 phosphate buffered saline 1:99 + 20 \LL formic acid + 5 mL cyclohexane: ethyl acetate 8:2, extract. Remove the organic layer and evaporate it, reconstitute the residue with 500 JULL mobile phase, inject a 10 jxL aliquot. HPLC VARIABLES Column: 250 X 4.6 C6H5-1252N (Senshu Sci.) Mobile phase: MeCN: 10 mM pH 3.3 tartrate buffer:PIC-A 52:48:0.5 Flow rate: 1 Injection volume: 10 Detector: F ex 293 em 325 CHROMATOGRAM Retention time: 7.5

Internal standard: 4-(2,5-dimethylphenoxy)-2,2-dimethylbutanoic acid Limit of detection: 100 ng/mL

OTHER SUBSTANCES

Simultaneous: metabolites
KEYWORDS

plasma
REFERENCE
Nakagawa, A.; Shigeta, A.; Iwabuchi, H.; Horiguchi, M.; Nakamura, K.; Takahagi, H. Simultaneous determination of gemfibrozil and its metabolites in plasma and urine by a fully automated high performance liquid chromatographic system. Biomed.Chromatogr., 1991, 5, 6873

SAMPLE

Matrix: blood Sample preparation: 1 mL Serum H - 2 mL MeCN: glacial acetic acid 75:10, vortex thoroughly, centrifuge. Remove the supernatant and add it to 500 mg NaCl, vortex, centrifuge. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at room temperature, reconstitute the residue in 200 fxL mobile phase, inject a 20 |xL aliquot.
HPLCVARIABLES

Column: 5 mm i.d. juiBondapak C18 radial compression Mobile phase: MeOH: water: glacial acetic acid 75:24:1 Flow rate: 0.8-1.2 Injection volume: 20 Detector: UV 276
CHROMATOGRAM

Retention time: 9-10 Limit of quantitation: 1 |xg/mL

KEYWORDS

serum
REFERENCE
Forland, S.C; Chaplin, L.; Cutler, R.E. Assay of gemfibrozil in plasma by "high-performance" liquid chromatography. Clin.Chem., 1987, 33, 1938

SAMPLE

Matrix: blood Sample preparation: 500 |JLL Plasma + 20 JJLL 100 fxg/mL ibuprofen in MeCN.water 50: 50, acidify with 3 drops 1 M HCl, add 5 mL cyclohexane, shake mechanically for 20 min, centrifuge. Remove the organic layer and evaporate it to dryness under reduced pressure, reconstitute the residue in 50-200 |xL mobile phase, inject a 10-20 |xL aliquot.
HPLCVARIABLES

Column: 125 X 4.6 5 (xm Spherisorb ODS II Mobile phase: MeCN: water: phosphoric acid 50:50:0.2 Flow rate: 2 Injection volume: 10-20 Detector: UV 225
CHROMATOGRAM

Retention time: 8.8

Internal standard: ibuprofen (5.8) Limit of detection: 50 ng/mL OTHER SUBSTANCES Noninterfering: metabolites KEYWORDS plasma; pharmacokinetics REFERENCE
Hengy, H.; Kolle, E.U. Determination of gemfibrozil in plasma by high performance liquid chromatography. Arzneimittelforschung, 1985, 35, 1637-1639

SAMPLE Matrix: blood, tissue Sample preparation: Acidify plasma with 10 |xL 5 M orthophosphoric acid. Homogenize 1 g tissue with 2 mL ice-cold 10 mM pH 5.0 phosphate buffer containing 1.15% KCl. 1 mL Plasma or tissue homogenate + 5 mL MeCN: acetic acid 96:4, centrifuge at 1000 g for 5 min, discard the supernatant, wash the pellet with 5 mL diethyl ether, wash nine times with 5 mL 4% acetic acid in MeCN: 10 mM pH 5.0 phosphate buffer 2:1. Add 1 mL 1 M KOH and 50 \xL 5 [xg/mL flurbiprofen in 0.06% MeCN to the pellet, heat at 80 overnight, cool, add 500 jxL 4 M HCl, add 5 mL diethyl ether, shake horizontally at 80 oscillations/min for 15 min, centrifuge. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 250 |xL mobile phase, inject a 50 JULL aliquot. HPLCVARIABLES Column: 125 X 4 5 |xm LiChrosphere 60 C8 RP-select B Mobile phase: MeCN: water: acetic acid 51:48.5:0.5 Flow rate: 1 Injection volume: 50 Detector: F ex 284 em 316 CHROMATOGRAM Retention time: 9 Internal standard: flurbiprofen (5) Limit of quantitation: 25 ng/mL OTHER SUBSTANCES Extracted: metabolites, glucuronides KEYWORDS rat; plasma; liver; kidney; heart; only gemfibrozil bound to protein is determined by this method REFERENCE
Sallustio, B.C.; Foster, D.J.R. Reactivity of gemfibrozil l-O-pi-acyl glucuronide. Pharmacokinetics of covalently bound gemfibrozil-protein adducts in rats. Drug Metab.Dispos., 1995, 23, 892-899

SAMPLE Matrix: blood, urine Sample preparation: 1 mL Plasma + 250 JJIL 200 [LgImL IS in MeCN: water 5:95 + 1 mL 1 M HCl H - 10 mL diethyl ether, shake on a reciprocating shaker for 15 min, centrifuge at 700 g. Remove the organic layer and evaporate it to dryness under a stream of air at 55, reconstitute the residue in 500 |xL mobile phase, mix, inject a 40 |xL aliquot. (To hydrolyze conjugates mix 1 mL plasma or urine with 25 |xL glucuronidase/sulfatase (GIu-

sulase, DuPont), 1 mL water, and 1 mL 2 M pH 5.2 acetate buffer, heat at 37 overnight, add 2 mL 1 M HCl, proceed as above.)
HPLCVARIABLES

Column: 100 X 4.6 5 jim Partisil ODS-3 RAC II Mobile phase: MeCN: 7 mM phosphoric acid 45:55 Flow rate: 2 Injection volume: 40 Detector: UV 276
CHROMATOGRAM

Retention time: 10.4 Internal standard: 2,2'-dimethyl-5-(2,6-xylyloxy)valeric acid (7.4) Limit of quantitation: 500 ng/mL OTHER SUBSTANCES Extracted: metabolites
KEYWORDS

plasma; pharmacokinetics; also for whole blood

REFERENCE
Randinitis, E.J.; Parker, T.D.; Kinkel, A.W. Liquid chromatographic determination of gemfibrozil and its metabolite in plasma. J.Chromatogr., 1986, 383, 444-448

SAMPLE

Matrix: solutions Sample preparation: Inject a 10 (JLL aliquot of a 200 jig/mL solution in mobile phase.
HPLCVARIABLES

Column: 300 X 4 5 |mm Suplecosil LC-18 Mobile phase: MeOH: water: glacial acetic acid 80:20:1 Flow rate: 0.8 Injection volume: 10 Detector: UV 276
CHROMATOGRAM

Retention time: 14 Internal standard: 2,5-xylenol (5)

REFERENCE
Supelco Chromatography Products, Supleco, Inc., Bellefonte PA, 1996, p. 155

SAMPLE

Matrix: solutions Sample preparation: Inject a 10-100 \xh aliquot.

HPLCVARIABLES

Column: 50 X 4.6 5 yon Supelcosil LC-18-DB or 100 X 4.6 5 \xm Supelcosil LC-18-DB Mobile phase: MeOH: water: acetic acid 80:19:1 Flow rate: 1 Injection volume: 10-100 Detector: UV 276

REFERENCE
Luner, RE.; Babu, S.R.; Radebaugh, G.W. The effects of bile salts and lipids on the physicochemical behavior of gemfibrozil. Pharm.Res., 1994, 11, 1755-1760

SAMPLE

Matrix: urine Sample preparation: 0.5 mL Urine + 0.5 mL 10 |xg/mL IS in MeCN, vortex, centrifuge at 2000 g, inject a 10 |xL aliquot of the upper layer.
HPLCVARIABLES

Column: 150 X 4.6 YMC-A312 ODS (Yamamura Chemicals) Mobile phase: MeCN: 10 mM pH 4.7 acetate buffer 45:55 for 10.5 min then 80:20 Flow rate: 1 for 10.5 min then 2 mL/min Injection volume: 10 Detector: F ex 283 em 315 (for gemfibrozil) and ex 300 em 340 (for metabolites)
CHROMATOGRAM

Retention time: 16 Internal standard: 7-(2,5-dimethylphenoxy)-2,2-dimethylheptanoic acid Limit of detection: 100 ng/mL
OTHER SUBSTANCES

Simultaneous: metabolites
REFERENCE
Nakagawa, A.; Shigeta, A.; Iwabuchi, H.; Horiguchi, M.; Nakamura, K.; Takahagi, H. Simultaneous determination of gemfibrozil and its metabolites in plasma and urine by a fully automated high performance liquid chromatographic system. Biomed.Chromatogr., 1991, 5, 68-73