Digoxin

Molecular formula: C41H64O14 Molecular weight: 781.0 CAS Registry No.: 20830-75-5

SAMPLE Matrix: blood Sample preparation: Condition a 1 mL Cyclobond I p-cyclodextrin SPE cartridge (Astec) with 2 mL MeOH, 2 mL MeCN, 2 mL isopropanol, and 2 mL water (SPE cartridge A). Condition a 1 mL Cyclobond I p-cyclodextrin SPE cartridge (Astec) with 2 mL MeOH, 2 mL MeCN, and 2 mL dichloromethane (SPE cartridge B). Condition a 1 mL Bond Elut Cl SPE cartridge with 2 mL MeOH and 2 mL MeCN (SPE cartridge C). 1 mL Serum + 10 ng digitoxin + 1 mL water, add to SPE cartridge A, wash with 2 mL water, wash with 1 mL MeOH: 7.5 mM pH 7.0 potassium phosphate buffer 20:80, wash with 3 mL water, wash with 1 mL isopropanol: water 10:90, dry under vacuum for 5 min, wash with ten 100 |xL aliquots of dichloromethane, dry under vacuum for 5 min, elute with 1 mL isopropanol. Evaporate the eluate to dryness under a stream of nitrogen at room temperature, add 50 (JLL 10% 4-dimethylaminopyridine in MeCN then 50 |xL 4% 1-naphthoyl chloride in MeCN under nitrogen in a glove box (relative humidity <26%), mix thoroughly, heat at 50for 1 h, centrifuge briefly, evaporate under a stream of nitrogen, add 2 mL 5% pH 10.0 sodium bicarbonate solution, shake for 1 min, add 2 mL chloroform, shake, centrifuge. Remove the organic layer and wash it with 2 mL 5% sodium bicarbonate, wash twice with 2 mL portions of 50 mM HCl, evaporate to dryness under a stream of nitrogen at room temperature, reconstitute with 200 |JLL dichloromethane, add to SPE cartridge B, wash with eight 100 (xL aliquots of dichloromethane, elute with 1 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute with 250 |xL MeCN, add to SPE cartridge C, rinse container with 250 |xL MeCN, add rinse to the SPE cartridge, add 500 |JLL MeCN to the SPE cartridge. Collect all the eluates and evaporate them to dryness under a stream of nitrogen, reconstitute with mobile phase, inject an aliquot. (Purify 4-dimethylaminopyridine by passing a 30% solution in MeCN through a layer of silica gel covered with a layer of activated charcoal, evaporate the filtrate under reduced pressure, store the residue in a desiccator. Immerse glassware in sulfuric acid: nitric acid 80:20 for 24 h, wash with water, treat with 1% Surfasil (Pierce) in toluene, rinse with water, dry in an oven.) HPLCVARIABLES Guard column: 15 X 3.2 7 |xm silica (Applied Biosystems) Column: 150 X 4.6 3 jxm Spherisorb silica Mobile phase: Hexane: dichloromethane: MeCN: MeOH 36:6.3:5.4:0.2 Flow rate: 1.6 Injection volume: 20 Detector: F ex 217 em 340 CHROMATOGRAM Retention time: 10 Internal standard: digitoxin (9.5) Limit of detection: 0.25 ng/mL

OTHER SUBSTANCES Extracted: metabolites, digoxigenin, digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, dihydrodigoxin Noninterfering: acetaminophen, acetazolamide, acyclovir, albuterol, allopurinol, amiodarone, amitriptyline, amoxicillin, ampicillin, aspirin, atenolol, atropine, azathioprine, bumetanide, calcitriol, captopril, carbamazepine, cefazolin, cefoperazone, ceftazidime, ceftizoxime, cephalexin, chlordiazepoxide, ciprofioxacin, clavulanic acid, clindamycin, clonidine, clotrimazole, codeine, conjugated estrogens, cyclophosphamide, diazepam, diphenhydramine, dipyridamole, dobutamine, docusate sodium, dopamine, enalapril, erythromycin, famotidine, fluconazole, furosemide, gemfibrozil, gentamicin, glyburide, heparin, hydralazine, hydrochlorothiazide, ibuprofen, ipratropium bromide, isosorbide dinitrate, isradipine, labetalol, lidocaine, lorazepam, lovastatin, medroxyprogesterone acetate, meperidine, metoclopramide, metolazone, metoprolol, midazolam, minoxidil, morphine, nicotine, nifedipine, nitroglycerin, norepinephrine, nystatin, oxybutynin, oxycodone, pentoxiphylline, phenytoin, piroxicam, prednisone, procainamide, procaine, promethazine, propoxyphene, ranitidine, sotalol, spironolactone, sulbactam, sulfamethoxazole, sulfisoxazole, temazepam, tetracycline, timolol, tobramycin, triamcinolone acetonide, triamterene, trimethoprim, vancomycin, verapamil, warfarin KEYWORDS normal phase; derivatization; serum; SPE; pharmacokinetics REFERENCE
Tzou, M.-C; Sams, R.A.; Reuning, R.H. Specific and sensitive determination of digoxin and metabolites in human serum by high-performance liquid chromatography with cyclodextrin solid-phase extraction and precolumn fluorescence derivatization. J.Pharm.Biomed.Anal, 1995, 13, 1531-1540

SAMPLE Matrix: blood Sample preparation: 3 mL Serum + 20 |xL 8 |xg/mL IS in EtOH + 3 mL acetone, vortex for 20 s, centrifuge at 1000 g for 5 min, remove the supernatant and add it to 2 mL isooctane: dichloromethane 80:20, vortex for 1 min, centrifuge at 1000 g for 5 min. Remove the acetone/water layer and evaporate it to 3 mL under a stream of nitrogen at 37, add 10 mL dichloromethane: n-propanol 98:2, rotate for 10 min, centrifuge at 1000 gfor 5 min, repeat extraction, filter the organic layers and evaporate them to dryness under a stream of nitrogen at 37, reconstitute the residue in 100 |JLL MeOH: water 50:50, inject the whole amount. HPLCVARIABLES Guard column: 15 X 3.2 ODS (Brownlee) Column: 150 X 4.6 3 |xm Spherisorb ODS II Mobile phase: MeOH: EtOH: isopropanol:buffer 52:3:1:45 (Prepare buffer by mixing 12.5 mL 0.15% hydrogen peroxide in water with 500 mL 500 |xg/mL L-ascorbic acid in water, stir for 2 h. Prepare fresh each week.) Flow rate: 0.4 Injection volume: 100 Detector: F ex 360 (filter) em 425 (filter) following post-column reaction. The column effluent mixed with concentrated HCl pumped at 0.5 mL/min and flowed through a 20 m X 0.3 mm i.d. PTFE coil at 79 1 to the detector. (The flow of concentrated HCl was generated by displacing concentrated HCl from a pressure vessel with hexane. The hexane was pumped into the pressure vessel by an HPLC pump.) CHROMATOGRAM Retention time: 18.5 Internal standard: digitoxigenin (25.5) Limit of quantitation: 0.5 ng/mL

OTHER SUBSTANCES Simultaneous: digoxigenin, digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, dihydrodigoxigenin, dihydrodigoxin, furosemide, spironolactone Noninterfering: mexiletine, captopril, dipyridamole, disopyramide, procainamide, propafenone, quinidine, sulfamethoxazole, trimethoprim, verapamil KEYWORDS serum; post-column reaction REFERENCE
Embree, L.; McErlane, K.M. Development of a high-performance liquid chromatographic-post-cohimn fluorogenic assay for digoxin in serum. J.Chromatogr., 1989, 496, 321-334

SAMPLE Matrix: blood Sample preparation: Wash a C18 Sep-Pak with 24 mL MeOH then 24 mL water. Wash a Diol Sep-Pak with 6 mL MeOH. 300 |xL Serum + 25 \xh 23.86 nmol/L deslanoside + 25 |JLL 28.68 ixmol/L gitoxigenin, vortex, add 300 |JLL to the C18 Sep-Pak, wash with 1 mL water, 1 mL ice-cold 100 g/L ZnSO4, 1 mL 20 mL/L MeCN, 3 mL water, remove excess water by applying vacuum for several min. Elute the C18 Sep-Pak with 3 mL MeOH through the Diol Sep-Pak and collect the eluate. Dry the eluate under nitrogen at 37, reconstitute in 200 |xL mobile phase, vortex 30 s, centrifuge at 1100 g for 15 min, inject 185-195 |JLL. After each run clean column by injecting 200 JJLL MeOH + 1 mL THF. Immunoassay detection. Gitoxigenin elutes at 16 min as a marker. Collect deslanoside (1012 min), digoxin (24-30 min), and 1 min fractions on either side of digoxin (4 tubes). Dry under air at 25, reconstitute residue with 230 (JLL digoxin-free serum, vortex 20 s, centrifuge at 1100 g for 5 min, use 200 |xL. Determine digoxin by fluorescence polarization immunoassay in accordance with manufacturer's instructions. HPLCVARIABLES Column: 100 X 4.6 3 |xm ODS2 (Chromatography Sciences Co.) Mobile phase: THF: water 20.5:79.5 Flow rate: 0.6 Injection volume: 185-195 Detector: UV 218; Immunoassay CHROMATOGRAM Retention time: 25 Internal standard: deslanoside OTHER SUBSTANCES Noninterfering: dexamethasone, hydroxyprogesterone, methylprednisolone, progesterone Interfering: cortisone (with UV assay), fludrocortisone (with UV assay), prednisone (with UV assay) KEYWORDS serum REFERENCE
Stone, J.A.; Soldin, S. J. Improved liquid chromatographic/immunoassay of digoxin in serum. Clin.Chem., 1988, 34, 2547-2551

SAMPLE Matrix: blood Sample preparation: 3 mL Plasma + 3 mL acetone, vortex, centrifuge at 1000 g for 5 min, remove the supernatant and add it to 2 mL isooctane, vortex, centrifuge at 1000 g

for 5 min. Remove the acetone/water layer and evaporate it to 3 mL under a stream of nitrogen at 37, add 10 mL dichloromethane: n-propanol 98:2, rotate for 10 min, centrifuge at 1000 g for 5 min, filter the organic layer and evaporate it to dryness under a stream of nitrogen at 37, reconstitute the residue in 100 |xL MeOH: water 50:50, inject the whole amount. HPLCVARIABLES Guard column: 37 |xm ODS Column: 150 X 4.6 3 jxm Spherisorb ODS II Mobile phase: MeOH: EtOH: isopropanol: water 52:3:1:45 Flow rate: 0.3 Injection volume: 100 Detector: F ex 360 (filter) em 425 (filter) following post-column reaction. The column effluent mixed with the reagent and flowed through a 10 m X 0.3 mm i.d. knitted PTFE coil at 79 1 to the detector. The reagent was generated by mixing 1.1 mM hydrogen peroxide in 0.1% ascorbic acid pumped at 0.038 mL/min and concentrated HCl pumped at 0.192 mL/min and allowing this mixture to flow through a 2 m X 0.8 mm i.d. PTFE coil to the point where it mixed with the column effluent (J.Chromatogr. 1986, 377, 233). CHROMATOGRAM Retention time: 33 Internal standard: digitoxigenin (42) Limit of detection: 0.5 ng/mL OTHER SUBSTANCES Simultaneous: digoxigenin, digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, dihydrodigoxigenin, dihydrodigoxin, furosemide, spironolactone Noninterfering: captopril, dipyridamole, disopyramide, procainamide, propafenone, quinidine, sulfamethoxazole, trimethoprim, verapamil KEYWORDS plasma; post-column reaction; comparison with RIA REFERENCE
Kwong, E.; McErlane, K.M. Analysis of digoxin at therapeutic concentrations using high-performance liquid chromatography with post-column derivatization. J.Chromatogr., 1986, 381, 357363

SAMPLE Matrix: blood, perfusate Sample preparation: 200 |xL Plasma or perfusate + 20 |xL ethinyl estradiol solution + 5 mL dichloromethane, vortex, centrifuge for 10 min. Remove a 4.5 mL aliquot of the lower organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 200 jxL mobile phase, inject a 50 |xL aliquot. HPLCVARIABLES Column: LiChrocart 100 RP-18 Mobile phase: MeOH: isopropanol: dichloromethane: water 40:9:4:47 Flow rate: 1 Injection volume: 50 Detector: UV 220 CHROMATOGRAM Internal standard: ethinyl estradiol (17a-ethynylestradiol) KEYWORDS plasma; rat; pharmacokinetics

REFERENCE
Su, S.-F.; Huang, J.-D. Inhibition of the intestinal digoxin absorption and exsorption by quinidine. Drug Metab.Dispos., 1996, 24, 142-147

SAMPLE Matrix: blood, urine Sample preparation: Urine. Extract 20 mL urine with 20 mL dichloromethane containing 3% heptafluorobutanol for 15 min, centrifuge at 1000 g for 10 min. Remove 15 mL aqueous phase, extract with 15 mL dichloromethane containing 3% heptafluorobutanol for 15 min, centrifuge at 1000 g for 10 min. Combine 10 mL volumes of each organic phase, evaporate under nitrogen to about 0.5 mL, add 20 |xL 1-pentanol, evaporate to 20 jxL, dissolve residue in 250 |xL mobile phase, inject a 100 |xL aliquot. Plasma. 10 mL Plasma + 3 g sodium chloride, extract with 15 mL dichloromethane containing 3% heptafluorobutanol for 15 min, centrifuge at 1000 g for 10 min. Evaporate 10 mL organic phase under nitrogen to about 0.5 mL, add 25 |xL phosphate buffer, evaporate to 25 |xL, dissolve residue in mobile phase, inject whole amount. (Plasma extraction in Acta Pharmacol. Toxicol. (Copenh.) 1986, 59 (Suppl. 4), 1-62.) HPLCVARIABLES Column: 150 X 4.5 7 ixm LiChrosorb RP-8 Mobile phase: Isopropanol: pH 6.3 phosphate buffer (1=0.1) 16.5:83.5 Flow rate: 1 Injection volume: 100 Detector: UV 220 CHROMATOGRAM Retention time: 23 Limit of detection: 2 ng/mL KEYWORDS plasma REFERENCE
Eriksson, B.-M.; Tekensbergs, L.; Magnusson, J.-O.; Molin, L. Determination of tritiated digoxin and metabolites in urine by liquid chromatography. J.Chromatogr., 1981, 223, 401-408

SAMPLE Matrix: bulk Sample preparation: Dissolve a small amount in 200 JJLL dry pyridine, add 15 mg 3,5dinitrobenzoyl chloride, shake for 2 h, evaporate to dryness under nitrogen under reduced pressure. Reconstitute with 1.5 mL ethyl acetate, wash 4 times with 1 mL portions of 5% sodium bicarbonate containing 2.5 mg/mL 4-dimethylaminopyridine, wash 4 times with 1 mL portions of 1% HCl, wash 4 times with 1 mL portions of water, evaporate to dryness under a stream of nitrogen, reconstitute with mobile phase, inject an aliquot (J.Chromatogr.Sci. 1983, 21, 495). HPLCVARIABLES Guard column: 15 X 3.2 Brownlee ODS Column: 150 X 4.6 3 fim Spherisorb ODS II Mobile phase: MeOH: EtOH: MeCN: isopropanol: 100 mM pH 4.6 sodium acetate buffer 40:3:60:2:22

Flow rate: 1
Detector: UV 254; E, ESA Coulochem Model 5100A, Model 5020 guard cell -0.8 V (placed before the injector), Model 5010 dual-electrode analytical cell with glassy-carbon electrodes (-0.8 V first electrode, +0.8 V second electrode)

CHROMATOGRAM Retention time: 13 Limit of detection: 0.39 ng (electrochemical detection) OTHER SUBSTANCES Simultaneous: digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, digoxigenin, dihydrodigoxigenin, dihydrodigoxin KEYWORDS derivatization REFERENCE
Embree, L.; McErlane, K.M. Electrochemical detection of 3,5-dinitrobenzoyl derivatives of digoxin by high-performance liquid chromatography. J.Chromatogr., 1990, 526, 439-446

SAMPLE Matrix: bulk, formulations Sample preparation: Ampoules. Add the contents of 1 ampoule (2 mL) to 15 mL 2% sodium bicarbonate solution, extract 5 times with 10 mL portions of chloroform: isopropanol 60:40, wash each extract with the same 10 mL portion of water, wash with another 10 mL portion of water. Combine the organic layers and evaporate them to dryness, transfer the residue to another tube with two 1 mL portions of chloroform: pyridine 10:1, evaporate to dryness under reduced pressure at 50, add 200 (xL reagent, shake well, let stand at room temperature for 10 min, evaporate to dryness under reduced pressure at 50, flush the tube with a stream of air or nitrogen, add 2 mL 5% sodium carbonate solution containing 2.5 mg/mL 4-dimethylaminopyridine, shake or sonicate for 5 min, extract with 2 mL chloroform. Wash the extract with 2 mL 5% sodium bicarbonate solution, wash twice with 3 mL portions of 50 mM HCl containing 5% NaCl, inject a 20 \xL aliquot. Bulk. Prepare a solution in pyridine containing not more than 10 mg/mL. Add 150 JJLL reagent to 50 JJLL solution, shake well, let stand at room temperature for 10 min, evaporate to dryness under reduced pressure at 50, flush the tube with a stream of air or nitrogen, add 2 mL 5% sodium carbonate solution containing 2.5 mg/mL 4-dimethylaminopyridine, shake or sonicate for 5 min, extract with 2 mL chloroform. Wash the extract with 2 mL 5% sodium bicarbonate solution, wash twice with 3 mL portions of 50 mM HCl containing 5% NaCl, inject a 20 |JLL aliquot. (Prepare reagent fresh each day by dissolving 100 mg 4-nitrobenzoyl chloride in 1 mL pyridine with gentle warming.) HPLCVARIABLES Column: 200 X 3 5 |xm Merckosorb SI 60 Mobile phase: n-Hexane: chloroform: MeCN 30:10:9 Flow rate: 1.5 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 6 Limit of detection: 11 ng/mL (100 |xL injection) OTHER SUBSTANCES Simultaneous: diginatigenin, diginatin, digitoxigenin, digitoxin, digoxigenin, gitaloxigenin, gitaloxin, gitoxigenin, gitoxin, lanatoside A, lanatoside B, lanatoside C, lanatoside D, lanatoside E KEYWORDS ampoules; normal phase; derivatization

REFERENCE
Nachtmann, R; Spitzy, H.; Frei, R.W. Rapid and sensitive high-resolution procedure for digitalis glycoside analysis by derivatization liquid chromatography. J.Chromatogr., 1976, 122, 293-303

SAMPLE Matrix: feces, urine Sample preparation: Urine. Place 1 mL 100 ng/mL digitoxin in isopropanol in a tube and evaporate. Add 1 mL urine + 2 mL dichloromethane, shake by hand 4 times, centrifuge 1650 g. Remove organic layer and wash it twice with 2 mL 5% sodium bicarbonate solution, evaporate under nitrogen at 50. Add 25 mg 4-dimethylaminopyridine and 10 fxL 1-naphthoyl chloride, add 100 (JLL MeCN, vortex thoroughly, place in water bath at 50 for 1 h, centrifuge, evaporate at 50 under nitrogen. Add 2 mL 5% sodium bicarbonate solution, shake mechanically for 5 min, add 2 mL chloroform, shake by hand. Remove organic layer and wash it twice with 2 mL 5% sodium bicarbonate solution, wash three times with 0.05 M HCl containing 5% NaCl, evaporate chloroform, dissolve residue in mobile phase. Feces. Dilute 5:1 (v/w) with 5 \xg/mL clindamycin in water to stop bacterial metabolism, homogenize with mechanical shaking for 15 min. Evaporate 1 mL 100 ng/mL digitoxin in isopropanol into a tube, weigh ca. 1 g homogenate into the tube, add 1 mL water, vortex 30 s, shake 15 min, centrifuge 1 h. Pour off supernatant and extract it with 2 mL dichloromethane. Wash the extract twice with 2 mL 5% sodium bicarbonate solution, evaporate under nitrogen at 50. Add 25 mg 4-dimethylaminopyridine and 10 JJLL 1-naphthoyl chloride, add 100 |ULL MeCN, vortex thoroughly, place in water bath at 50 for 1 h, centrifuge, evaporate at 50 under nitrogen. Add 2 mL 5% sodium bicarbonate solution, shake mechanically for 5 min, add 2 mL chloroform, shake by hand. Remove organic layer and wash it twice with 2 mL 5% sodium bicarbonate solution, wash three times with 0.05 M HCl containing 5% NaCl, evaporate chloroform, dissolve residue in mobile phase. HPLCVARIABLES Column: 150 X 4.6 3 |xm Adsorbosphere SI Mobile phase: Hexane: dichloromethane: MeCN 6:1:1 Flow rate: 1.8-2 Injection volume: 20-175 Detector: F ex 217 em 340 cut-off filter (372 nm max) CHROMATOGRAM Retention time: 9.4 Internal standard: digitoxin (8.1) Limit of detection: 5 ng/mL (urine); 50 ng/g (feces) OTHER SUBSTANCES Simultaneous: metabolites KEYWORDS normal phase REFERENCE
Shepard, TA.; Hui, J.; Chandrasekaran, A.; Sams, R.A.; Reuning, R.H.; Robertson, L.W.; Caldwell, J.H.; Donnerberg, R.L. Digoxin and metabolites in urine and feces: a fluorescence derivatization-highperformance liquid chromatographic technique. J.Chromatogr., 1986, 380, 89-98

SAMPLE Matrix: formulations Sample preparation: Dilute a 1 mL sample to 10 mL with mobile phase, inject an aliquot. HPLC VARIABLES Column: 250 X 4.6 10 |xm Partisil ODS III C18

Mobile phase: MeCN: water: phosphoric acid 35:65:0.1 Flow rate: 2 Injection volume: 20 Detector: UV 220 CHROMATOGRAM Retention time: 7 OTHER SUBSTANCES Noninterfering: amrinone KEYWORDS injections; stability-indicating; 5% dextrose; 0.45% NaCl REFERENCE
Riley, CM.; Junkin, R Stability of amrinone and digoxin, procainamide hydrochloride, propranolol hydrochloride, sodium bicarbonate, potassium chloride, or verapamil hydrochloride in intravenous admixtures. Am.J.Hosp.Pharm., 1991, 48, 1245-1252

SAMPLE Matrix: formulations Sample preparation: One tablet (0.25 mg digoxin) H - 5 mL acetone:ethanol 9:1 containing 0.11826 mg dexamethasone, sonicate 5 min, centrifuge at 1400 g for 5 min, evaporate supernatant under vacuum, dissolve residue in 100 jxL MeOH, inject 0.2 JJLL aliquots. HPLCVARIABLES Column: 95 X 0.5 Japan Spectroscopic SC-Ol (5 jxm octadecylsilyl silica in a PTFE tube) Mobile phase: MeCN: water 28:72 Flow rate: 0.008 Injection volume: 0.2 Detector: UV 220 CHROMATOGRAM Retention time: 10 Internal standard: dexamethasone (14) OTHER SUBSTANCES Simultaneous: digoxigenin, digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, dimethyldigoxin, p-methyldigoxin KEYWORDS tablets; microbore REFERENCE
Fujii, Y.; Ikeda, Y; Yamazaki, M. High-performance liquid chromatographic determination of secondary cardiac glycosides in Digitalis purpurea leaves. J.Chromatogr., 1989, 479, 319-325

SAMPLE Matrix: formulations Sample preparation: 1 mL Sample + 9 mL mobile phase, mix, inject an aliquot. HPLCVARIABLES Column: 150 X 4.6 5 fxm Zorbax ODS Mobile phase: MeCN:phosphoric acid:water 26:0.1:58, adjusted to pH 6.5 with 10 M NaOH

Flow rate: 2 Injection volume: 20 Detector: UV 218 CHROMATOGRAM Retention time: 6 OTHER SUBSTANCES Simultaneous: digoxigenin, digoxigenin didigitoxoside, digoxigenin monodigitoxoside

Noninterfering: milrinone
KEYWORDS stability-indicating; 5% dextrose; injections REFERENCE
Riley, CM. Stability of milrinone and digoxin, furosemide, procainamide hydrochloride, propranolol hydrochloride, quinidine gluconate, or verapamil hydrochloride in 5% dextrose injection. Am.J.Hosp.Pharm., 1988, 45, 2079-2091

SAMPLE Matrix: formulations Sample preparation: Directly inject a 20 jxL aliquot of a 250 |Jig/mL digoxin injection. HPLCVARIABLES Column: 300 X 3.9 10 jxm ixBondapak C18 Mobile phase: MeCN: water 29:71 Flow rate: 2 Injection volume: 20 Detector: UV 218 CHROMATOGRAM Retention time: 9 OTHER SUBSTANCES Simultaneous: mercaptobenzothiazole KEYWORDS injections REFERENCE
Reepmeyer, J.C.; Juhl, Y.H. Contamination of injectable solutions with 2-mercaptobenzothiazole leached from rubber closures. J.Pharm.Sci., 1983, 72, 1302-1305

SAMPLE Matrix: solutions Sample preparation: Inject a 10 |JLL aliquot of a solution in mobile phase. HPLCVARIABLES Column: 250 X 4.6 Deltabond C18 (Keystone) Mobile phase: Gradient. MeCN: water from 10:90 to 45:55 over 8 min.

Flow rate: 1.3 Injection volume: 10

Detector: E, Dionex pulsed electrochemical detector, integrated amperometry mode, 1.4 mm gold working electrode with 0.005 inch gasket, El +0.07 V, t l 400 ms, E2 +0.70 V, t2 120 ms, E3 1.00 V, t3 300 ms, stainless steel counter electrode, Ag/AgCl reference

electrode, following post-column reaction. The column effluent mixed with 1 M NaOH pumped at 0.5 mL/min and the mixture flowed through a 500 |xL reaction coil (Dionex) to the detector. CHROMATOGRAM Retention time: 9 Limit of detection: 230 ng/mL OTHER SUBSTANCES Simultaneous: digitoxigenin, digitoxigenin bisdigitoxoside, digitoxigenin monodigitoxoside, digitoxin, digoxigenin, digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside KEYWORDS post-column reaction REFERENCE
Kelly, K.L.; Kimball, B.A.; Johnston, J.J. Quantitation of digitoxin, digoxin, and their metabolites by high-performance liquid chromatography using pulsed amperometric detection. J.Chromatogr.A, 1995,711,289-295

SAMPLE Matrix: solutions HPLC VARIABLES Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine 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. Column temperature: 30 Flow rate: 2 Detector: UV 210 OTHER SUBSTANCES Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, 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, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephen-

termine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, 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, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triproilidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine REFERENCE
Hill, D.W.; Kind, A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs. J.Anal.Toxicol., 1994, 18, 233-242

SAMPLE Matrix: solutions HPLCVARIABLES Guard column: 37 |xm ODS Column: 150 X 4.6 3 |xm Spherisorb ODS II Mobile phase: MeOH: EtOH: isopropanol: water 52:3:1:45

Flow rate: 0.3

Detector: F ex 360 (filter) em 425 (filter) following post-column reaction. The column effluent mixed with the reagent and flowed through a 10 m X 0.3 mm i.d. knitted PTFE coil at 79 1 to the detector. The reagent was generated by mixing 1.1 mM hydrogen peroxide in 0.1% ascorbic acid pumped at 0.038 mL/min and concentrated HCl pumped at 0.192 mL/min and allowing this mixture to flow through a 2 m X 0.8 mm i.d. PTFE coil to the point where it mixed with the column effluent. CHROMATOGRAM Retention time: 33 OTHER SUBSTANCES Simultaneous: digitoxigenin, digoxigenin, digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, dihydrodigoxigenin, dihydrodigoxin, furosemide, spironolactone KEYWORDS post-column reaction REFERENCE
Kwong, E.; McErlane, K.M. Development of a high-performance liquid chromatographic assay for digoxin using post-column fluorogenic derivatization. J.Chromatogr., 1986, 377, 233-242

SAMPLE Matrix: urine Sample preparation: 10 mL Urine + 2 mL 1 M HCl (check pH is 1-2), heat 37 for 3 h, add 5 mL pH 6.5 phosphate buffer, add 2 mL 1 M NaOH (check pH is 6.5-7.0). Add to a 20 cm Extrelut SPE column, rinse flask with 3 mL water, add rinsings to column, dry for 15 min, elute with 40 mL dichloromethane, evaporate eluent to dryness, dry over concentrated sulfuric acid. Prepare a 100 mg/mL solution of 4-nitrobenzoyl chloride (4-NBP) in dry pyridine with gentle heating. Use immediately. Dissolve residue from column in 30 |xL dry pyridine, add 20 JJLL 2 mg/mL digitoxigenin in pyridine, add 300 JJLL 4-NBP solution, shake well. Heat at 70 for 1 h, add 2 mL 5% sodium bicarbonate, shake until precipitate has dissolved, add 2 mL chloroform, shake, centrifuge, repeat extraction twice. Combine chloroform layers, wash three times with 2 mL 1 M HCl, inject an aliquot of chloroform solution directly. HPLCVARIABLES Column: 200 X 4 Hibar 5 |xm Lichrosorb Si 60 Mobile phase: n-Hexane: dichloromethane: methanol 82.9:14.2:2.9 Flow rate: 1.2 Injection volume: 20 Detector: UV 258 CHROMATOGRAM Retention time: 12 Internal standard: digitoxigenin (8) Limit of detection: 1 (xg/mL KEYWORDS normal phase; derivatization; SPE; digoxin is hydrolysed to digoxigenin and determined as its 4-NBP derivative REFERENCE
Jakobsen, P.; Waldorff, S. Determination of digoxin, digoxigenin and dihydrodigoxigenin in urine by extraction, derivatization and high-performance liquid chromatography. J.Chromatogr., 1986, 382, 349-354

SAMPLE Matrix: urine Sample preparation: 10 mL Urine + 0.5 mL 20 |xg/mL digitoxigenin in dichloromethane + 20 mL dichloromethane, shake for 15 min, centrifuge for 20 min. Remove the organic phase and add it to 15 mL 5% sodium bicarbonate, shake for 15 min, centrifuge for 20 min. Remove the organic phase and evaporate it to dryness at 50 under a stream of nitrogen, add 200 |xL derivatizing solution to the residue, shake gently at room temperature for 10 min, evaporate to dryness under a stream of nitrogen at 50, add 2 mL 2 mg/mL 4-dimethylaminopyridine in 5% sodium bicarbonate, shake for 5 min, add 1 mL chloroform, rock on an Aliquot Mixer. Remove the organic phase and add it to 2 mL 5% sodium bicarbonate solution, mix for 2 min. Remove the organic phase and add it to 3 mL 50 mM HCl containing 5% NaCl, mix for 2 min. Remove the organic phase and repeat the acid wash 3 more times, inject a 100 |xL aliquot of the organic phase. (The derivatizing solution was 85 mg/mL 3,5-dinitrobenzoyl chloride in pyridine, prepared with gentle warming to help the solid dissolve.) HPLC VARIABLES Column: 250 X 4.6 10 |xm Partisil 10 Mobile phase: Hexane: dichloromethane: MeCN 60:20:20 Flow rate: 1.8

Injection volume: 100 Detector: UV 254 CHROMATOGRAM Retention time: 54 Internal standard: digitoxigenin (17) Limit of detection: 100 ng/mL OTHER SUBSTANCES Simultaneous: digoxigenin, digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside KEYWORDS normal phase REFERENCE
Bockbrader, H.N.; Reuning, R.H. Digoxin and metabolites in urine: A derivatiz at ion-high-performance liquid chromatographic method capable of quantitating individual epimers of dihydrodigoxin. J.Chromatogr., 1984, 310, 85-95

SAMPLE Matrix: urine Sample preparation: Extract 20 mL urine with 20 mL dichloromethane containing 3% heptafluorobutanol for 15 min, centrifuge at 1000 g for 10 min. Remove 15 mL aqueous phase, extract with 15 mL dichloromethane containing 3% heptafluorobutanol for 15 min, centrifuge at 1000 g for 10 min. Combine 10 mL volumes of each organic phase, evaporate
under nitrogen to about 0.5 mL, add 20 JJLL 1-pentanol, evaporate to 20 jxL, dissolve residue in 250 JJLL mobile phase, inject a 100 jxL aliquot.

HPLCVARIABLES Column: 150 X 4.5 5 |xm LiChrosorb SI 60 Mobile phase: n-Heptane: 1-pentanol: MeCN: water 64:26:9:1 Flow rate: 1.5 Injection volume: 100 Detector: UV 220 CHROMATOGRAM Retention time: 15 Limit of detection: 10 ng/mL KEYWORDS normal phase REFERENCE
Eriksson, B.-M.; Tekensbergs, L.; Magnusson, J.-O.; Molin, L. Determination of tritiated digoxin and metabolites in urine by liquid chromatography. J.Chromatogr., 1981, 223, 401-408

ANNOTATED BIBLIOGRAPHY

Ikeda, Y; Fujii, Y; Nakaya, L; Yamazaki, M. Quantitative HPLC analysis of cardiac glycosides in Digitalis purpurea leaves. J.Nat.Prod., 1995, 58, 897-901 Hui, J.; Geraets, D.R.; Chandrasekaran, A.; Wang, Y.-M.C; Caldwell, J.H.; Robertson, L.W.; Donnerberg, R.L.; Reuning, R.H. Digoxin disposition in elderly humans with hypochlorhydria. J.CHn.Pharmacol., 1994, 34, 734-741 [urine; feces; derivatization; fluorescence detection; normal phase; digitoxin (IS); extracted metabolites; LOD 5 ng/mL] Oosterkamp, A. J.; Irth, H.; Beth, M.; Unger, K.K.; Tjaden, U.R.; van de Greef, J. Bioanalysis of digoxin and its metabolites using direct serum injection combined with liquid chromatography and on-line

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immunochemical detection. J.Chromatogr.B, 1994, 653, 55-61 [LOD 160 pg/mL; serum; columnswitching; post-column reaction; fluorescence detection; extracted metabolites; pharmacokinetics] Ikeda, Y; Fujii, Y.; Yamazaki, M. Determination of lanatoside C and digoxin in Digitalis lanataby HPLC and its application to analysis of the fermented leaf powder. J.Nat.Prod., 1992, 55, 748-752 Nakashima, H.; Tsutsumi, K.; Hashiguchi, M.; Kumagai, Y; Ebihara, A. Determination of ^-methyldigoxin and its metabolites by high-performance liquid chromatography and fluorescence polarization immunoassay. J.Chromatogr., 1989, 489, 425-431 Fujii, Y; Ikeda, Y; Yamazaki, M. Micro high-performance liquid chromatographic determination of cardiac glycosides in (3-methyldigoxin and digoxin tablets. J.Chromatogr., 1988, 448, 157-164 Reh, E. Determination of digoxin in serum by on-line immunoadsorptive clean-up high-performance liquid chromatographic separation and fluorescence-reaction detection. J.Chromatogr., 1988, 433, 119-130 [serum; column-switching; LOD 300 pg/mL; post-column reaction; fluorescence detection] Desta, B. Separation of digoxin from dihydrodigoxin and the other metabolites by high-performance liquid chromatography with post-column derivatization. J.Chromatogr., 1987, 421, 381-386 [postcolumn reaction; fluorescence detection; simultaneous metabolites] Plum, J.; Daldrup, T. Detection of digoxin, digitoxin, their cardioactive metabolites and derivatives by high-performance liquid chromatography and high-performance liquid chromatography-radioimmunoassay. J.Chromatogr., 1986, 377, 221-231 [gradient; tissue; extracted metabolites; SPE] Vetticaden, S.J.; Barr, W.H.; Beightol, L.A. Improved method for assaying digoxin in serum using highperformance liquid chromatography-radioimmunoassay. J.Chromatogr, 1986, 383, 187-193 [serum; dog; RIA detection] de Jong, H.C; Voogt, W.H.; Bos, P.; Frei, R.W. Tensammetric detection in high performance liquid chromatography. Application to lynestrenol and some cardiac glycosides. J.Liq.Chromatogr., 1983, 6, 1745-1758 [also lynestrenol; electrochemical detection] Gandelman, M.S.; Birks, J.W. Liquid chromatographic detection of cardiac glycosides, saccharides and hydrocortisone based on the photoreduction of 2-tert-butylanthraquinone. Anal. Chim.Ada, 1983, 155, 159-171 [post-column reaction; simultaneous diginatin; also hydrocortisone; LOD 2 ng] Wagner, J.G.; Dick, M.; Behrendt, D.M.; Lockwood, G.F.; Sakmar, E.; Hees, P. Determination of myocardial and serum digoxin concentrations in children by specific and nonspecific assay methods. Clin.Pharmacol.Ther, 1983, 33, 577-584 [heart; tissue; serum; ethoxzolamide (IS); RIA detection] Desta, B.; Kwong, E.; McErlane, K.M. Separation of digoxin, digitoxin and their potential metabolites, impurities or degradation products by high-performance liquid chromatography. J.Chromatogr., 1982,240, 137-143 Gault, H.; Kalra, J.; Ahmed, M.; Kepkay, D.; Longerich, L.; Barrowman, J. Influence of gastric pH on digoxin bio transformation. II. Extractable urinary metabolites. Clin.Pharmacol.Ther., 1981, 29, 181-190 [urine; radioactivity detection; tritium labeled] Loo, J.C.K.; McGilveray, I.J.; Jordan, N. The estimation of serum digoxin by combined HPLC separation and radioimmunological assay. J.Liq.Chromatogr, 1981, 4, 879-886 [serum; RIAdetection; extracted metabolites]