Doxycycline

Molecular formula: C22H24N2O8 Molecular weight: 444.4 CAS Registry No.: 564-25-0,17086-28-1 (monohydrate), 24390-14-5 (HCI monohydrate), 83038-87-3 (fosfatex), 24390-14-5 (hyclate)

SAMPLE Matrix: blood Sample preparation: 100 ^xL Serum + 50 JJLL 6% aqueous ascorbic acid + 50 ng demeclocycline in MeOH + 400 |xL buffer, vortex 30 s, add 3 mL ethyl acetate, vortex 5 min, centrifuge at 3000 rpm for 6 min. Remove organic layer and add it to 100 fxL 0.2% ascorbic acid in MeOH. Evaporate to dryness at 20 in a vortex evaporator, dissolve residue in 100 |xL mobile phase, inject entire amount. (Buffer was 2 M NaH2PO4 and 2 M Na2SO3, pH 6.1.) HPLCVARIABLES Guard column: 4 \xm Nova-Pak C18 Guard-Pak Column: 150 X 4.6 5 |xm Ultrabase C18 Mobile phase: MeCN: water adjusted to pH 2.5 with phosphoric acid 28:72 Flow rate: 1 Injection volume: 100 Detector: UV 350 CHROMATOGRAM Retention time: 4.2 Internal standard: demeclocycline (2.7) Limit of quantitation: 20 ng/mL KEYWORDS serum REFERENCE
Gastearena, L; Dios-Vieitez, M.C.; Segura, E.; Gofii, M.M.; Renedo, M.J.; Fos, D. Determination of doxycycline in small serum samples by liquid chromatography Application to pharmacokinetical studies on small laboratory animals. Chromatographia, 1993, 35, 524-526

SAMPLE Matrix: blood Sample preparation: 100 |JLL Plasma + 20 |JLL trifluoroacetic acid, mix 30 s in a whirl mixer, centrifuge at 5400 g for 5 min, inject supernatant (80 (xL). HPLCVARIABLES Guard column: 10 |xm Waters RP phenyl Column: 125 X 4.6 10 ^m Waters RP phenyl Mobile phase: MeCN: 10 mM phosphoric acid 30:70 Flow rate: 2 Injection volume: 80 Detector: UV 270 CHROMATOGRAM Retention time: 2.2 Limit of detection: 15 ng/mL

KEYWORDS plasma REFERENCE

Kramer-Horaczynska, F. High-performance liquid chromatographic procedures for the quantitative analysis of 15 tetracycline derivatives in small blood samples. J.Chromatogr.Sci., 1991, 29, 107-113

SAMPLE Matrix: blood, urine Sample preparation: Serum. 0.5 mL Serum + 0.5 mL MeCN:85% phosphoric acid: water 20:2:78, vortex, filter (10 000 or 30 000 Da cutoff) by centrifuging at 2200 g for 30 min, inject 10 |xL aliquot of filtrate. Urine. Dilute urine 5 to 10 times with MeCN: 85% phosphoric acid:water 20:1.7:78.3, vortex, filter (10 000 or 30 000 Da cutoff) by centrifuging at 2200 g for 30 min, inject 10 |xL aliquot of filtrate. HPLCVARIABLES Column: 220 X 4.6 phenyl Mobile phase: MeOH:MeCN:triethylamine:phosphoric acid:80 mM pH 2.4 sodium phosphate buffer 10:1.5:0.5:1.7:86.3 Column temperature: 50 Flow rate: 0.6-0.8 Injection volume: 10 Detector: UV 268; UV 345 CHROMATOGRAM Retention time: 8 Limit of detection: <10 ng/mL KEYWORDS serum; cow REFERENCE
Riond, J.L.; Hedeen, K.M.; Tyczkowska, K.; Riviere, J.E. Determination of doxycycline in bovine tissues and body fluids by high-performance liquid chromatography using photodiode array ultraviolet-visible detection. J.Pharm.ScL, 1989, 78, 44-47

SAMPLE Matrix: blood, urine Sample preparation: Serum. Condition a Bond-Elut C18 SPE cartridge with 1 volume MeOH and 2 volumes water. 1 mL Serum + 5 mL buffer, add to SPE cartridge, wash with 10 mL water, elute with 10 mL 10 mM phosphoric acid in MeCN. Evaporate eluate to dryness at 50 under a stream of nitrogen and resuspend residue in 1 mL water. Centrifuge at 10000 g for 1 min, inject 100 |xL aliquot. Urine. Activate a Bond-Elut C18 cartridge with 1 volume MeOH and 2 volumes water. 1 mL Urine + 5 mL buffer, add to cartridge, wash with 10 mL MeCN, elute with 10 mL 10 mM phosphoric acid in MeCN. Evaporate eluate to dryness at 50 under a stream of nitrogen and resuspend residue in 1 mL water. Centrifuge at 10 000 g for 1 min, inject 100 |xL aliquot. (Buffer was 0.1 M citric acid.0.2 M Na2HPO4 61.4:38.6 (Mcllvaines buffer) containing 0.1 M disodium EDTA.) HPLCVARIABLES Guard column: LiChrosorb RP-18 Column: 150 X 3.9 4 |xm Nova-Pak C18 Mobile phase: MeCN.acetic acid: 100 mM KH2PO4 75:150:125 (serum) or 65:150:125 (urine) Flow rate: 1

Injection volume: 100 Detector: UV 340 CHROMATOGRAM Retention time: 4 Limit of detection: 25 ng/mL KEYWORDS serum; SPE; protect from light with amber glassware REFERENCE
Sheridan, M.E.; Clarke, G.S. Improved high-performance liquid chromatographic determination of doxycycline in serum and urine using solid-phase extraction columns. J.Chromatogr., 1988, 434, 253258

SAMPLE Matrix: blood, urine Sample preparation: Serum. 500 |JLL Serum + 50 |xL 6% ascorbic acid in water + 50 |xL demeclocycline in MeOH/100 mM HCl + I m L buffer, mix for 30 s, add 6 mL ethyl acetate, rotate for 10 min, centrifuge at 3000 rpm for 6 min. Remove the organic layer and add it to 100 |xL 0.2% ascorbic acid in MeOH, evaporate to dryness under vacuum while vortexing, reconstitute the residue in 200 |xL mobile phase, mix, filter, keep in ice, inject a 20 |JLL aliquot. Urine. 100 |xL Urine + 50 |JLL 6% ascorbic acid in water + 50 |JLL demeclocycline in MeOH/100 mM HCl + 400 |JLL buffer, mix for 30 s, add 3 mL ethyl acetate, rotate for 10 min, centrifuge at 3000 rpm for 6 min. Remove the organic layer and add it to 100 |xL 0.2% ascorbic acid in MeOH, evaporate to dryness under vacuum while vortexing, reconstitute the residue in 200 |xL mobile phase, mix, filter, keep in ice, inject a 20 |xL aliquot. (Buffer was 27.6 g NaH2PO4 + 25.2 g sodium sulfite in 100 mL water, pH 6.1.) HPLCVARIABLES Column: 100 X 2 5 |xm Lichrosorb RP8 Mobile phase: MeCN: 100 mM citric acid 24:76 Flow rate: 0.5 Injection volume: 20 Detector: UV 350 CHROMATOGRAM Retention time: 9 Internal standard: demeclocycline (4) Limit of detection: 50 ng/mL OTHER SUBSTANCES Extracted: chlortetracycline, methacycline, oxytetracycline, tetracycline KEYWORDS serum REFERENCE
De Leenheer, A.R; Nelis, H.J.C.F. Doxycycline determination in human serum and urine by high-performance liquid chromatography. J.Pharm.ScL, 1979, 68, 999-1002

SAMPLE Matrix: bulk, formulations Sample preparation: Bulk. Prepare a 10-100 |xg/mL solution in buffer, inject an aliquot. Capsules, tablets. Prepare a 1 mg/mL solution of capsule contents or crushed tablets in

buffer, sonicate for 10 min, filter (0.45 ixm), dilute with buffer, inject an aliquot. (Buffer was 20 mM sodium perchlorate adjusted to pH 2.0 with perchloric acid.) HPLCVARIABLES Column: 250 X 4.6 5 jxm 100 A PLRP-S polystyrene-divinylbenzene (Polymer Laboratories) Mobile phase: MeCN: buffer 25:75 (Buffer was 20 mM sodium perchlorate adjusted to pH 2.0 with perchloric acid.) Flow rate: 1 Detector: UV 280 CHROMATOGRAM Retention time: 40 OTHER SUBSTANCES Simultaneous: impurities KEYWORDS capsules; tablets REFERENCE
Bryan, P.D.; Stewart, J.T. Chromatographic analysis of selected tetracyclines from dosage forms and bulk drug substance using polymeric columns with acidic mobile phases. J.Pharm.Biomed.Anal., 1994, 12, 675-692

SAMPLE Matrix: cell suspensions Sample preparation: 300 |xL Cell suspension + 300 |xL MeCN, vortex, centrifuge, inject a 10 jxL aliquot. HPLCVARIABLES Column: 125 X 4 Nucleosil 100 5CN Mobile phase: MeCN: THF: phosphate/citrate buffer 10:10:80 Injection volume: 10 Detector: UV 350 CHROMATOGRAM Retention time: 2.1 REFERENCE
Kersten, A.; Poitschek, C; Rauch, S.; Aberer, E. Effects of penicillin, ceftriaxone, and doxycycline on morphology of Borrelia burgdorferi. Antimicrob.Agents Chemother., 1995, 39, 1127-1133

SAMPLE Matrix: food Sample preparation: Condition a 100 mg Baker 10 C18 SPE cartridge by washing with MeOH, water, and 10 mL saturated aqueous Na2EDTA. Dissolve 5 g honey in 20 mL 100 mM pH 4.0 Na2EDTA-McIlvaine buffer, filter, apply to the SPE cartridge, wash with 20 mL water, air dry under vacuum for 5 min. Condition a Baker 10 COOH cartridge with ethyl acetate. Elute contents of C18 cartridge onto COOH cartridge with 50 mL ethyl acetate. Wash COOH cartridge with 10 mL MeOH, elute with 10 mL mobile phase, inject 100 |JLL aliquot. HPLCVARIABLES Column: 250 X 4.6 5 fxm Bakerbond C8 Mobile phase: MeOH: MeCN .10 mM aqueous oxalic acid 1:1.5:3

Flow rate: 1 Injection volume: 100 Detector: UV 350

CHROMATOGRAM

Retention time: 6 Limit of detection: 0.05 ppm

OTHER SUBSTANCES

Extracted: chlortetracycline, oxytetracycline, tetracycline

KEYWORDS

honey; SPE
REFERENCE
Oka, H.; Ikai, Y; Kawamura, N.; Uno, K.; Yamada, M.; Harada, K.; Uchiyama, M.; Asukabe, H., Mori, Y; Suzuki, M. Improvement of chemical analysis of antibiotics. IX. A simple method for residual tetracyclines analysis in honey using a tandem cartridge clean-up system. J.Chromatogr., 1987, 389, 417-426

SAMPLE

Matrix: food Sample preparation: Condition a 500 mg Baker-10 C18 SPE cartridge with 10 mL MeOH, 10 mL water, and 10 mL saturated aqueous disodium EDTA. Condition a 500 mg Baker10 COOH cartridge with MeOH: ethyl acetate 10:90. Dissolve 25 g honey in 50 mL 100 mM pH 4.0 disodium EDTA-Mcllvaine buffer, filter. Add the filtrate to the C18 SPE cartridge, wash with 20 mL water, wash with 400 \xL ethyl acetate, air dry under vacuum for 5 min, elute with 50 mL MeOH: ethyl acetate 10:90. Add a 5 mL aliquot to the COOH SPE cartridge, wash with 5 mL MeOH (?), elute with 10 mL mobile phase, inject a 100 |JLL aliquot.
HPLCVARIABLES

Column: 75 X 4.6 3 |xm Chemcosorb 3C8 (Chemco) Mobile phase: MeCN:MeOH: 10 mM aqueous oxalic acid 3:2:16, pH 3.0 Flow rate: 1 Injection volume: 100 Detector: UV 350
CHROMATOGRAM

Retention time: 9 Limit of detection: 0.1 ppm

OTHER SUBSTANCES

Extracted: chlortetracycline, demeclocycline (demethylchlortetracycline), methacycline, minocycline, oxytetracycline, tetracycline

KEYWORDS

honey; SPE
REFERENCE
Oka, H.; Ikai, Y.; Kawamura, N.; Uno, K.; Yamada, M.; Harada, K.; Suzuki, M. Improvement of chemical analysis of antibiotics. XIL Simultaneous analysis of seven tetracyclines in honey. J.Chromatogr., 1987, 400, 253-261

SAMPLE

Matrix: formulations

HPLCVARIABLES Column: 250 X 4.6 5 |xm Bakerbond phenylethyl Mobile phase: MeOHrIOO mM NaH2PO4 70:30 Flow rate: 0.8 Detector: UV 280 CHROMATOGRAM Retention time: 5.25 (doxycline hyelate) KEYWORDS injections; saline; water; stability-indicating REFERENCE
Stiles, M.L.; Allen, L.V., Jr.; Prince, S. J. Stability of various antibiotics kept in an insulated poucb during administration via portable infusion pump. Am.J.Health-Syst.Pharm., 1995, 52, 70-74

SAMPLE Matrix: formulations Sample preparation: Dissolve ointment in petroleum ether, add an equal volume of EtOH-.water 70:30, dilute with MeOH to 100 |xg/mL, inject a 10 |JLL aliquot. HPLCVARIABLES Column: 300 X 3.9 10 jxm LiChrosorb Si-60 Mobile phase: MeOH: water 5:95 containing 1.3 mM disodium citrate, 1 mM tetrabutylammonium bromide, 1.1 mM citric acid, and 8 mM EDTA. Flow rate: 1 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: k' 0.56 OTHER SUBSTANCES Simultaneous: anhydrotetracycline, chlortetracycline, demeclocycline, epianhydrotetracycline, oxytetracycline, quatrimycin, rolitetracycline, tetracycline KEYWORDS ointment REFERENCE
Lingeman, H.; van Minister, H.A.; Beynen, J.H.; Underberg, W.J.; Hulshoff, A. High-performance liquid chromatographic analysis of basic compounds on non-modified silica gel and aluminium oxide with aqueous solvent mixtures. J.Chromatogr., 1986, 352, 261-274

SAMPLE Matrix: milk Sample preparation: Prepare a column as follows. Swirl Chelating Sepharose Fast Flow resin (Pharmacia) in its bottle, add it to a polypropylene column to give a bed volume of 1.0-1.2 mL, wash 3 times with 2 mL portions of water, wash with 2 mL 10 mM copper sulfate, wash with two 2 mL portions of water. Centrifuge 5 mL milk at 10 at 1500 g for 15 min, remove the lower layer and add it to 10 mL succinate buffer, mix, centrifuge at 1500 g for 30 min, add the supernatant to the column. Wash with 2 mL succinate buffer, wash with 2 mL water, wash with 2 mL MeOH, wash with 2 mL water, wash with 700 IxL citrate/phosphate buffer (be careful not to disturb bed), elute with 2.5 mL citrate/phosphate buffer (column is white and eluate is blue). Filter (Amicon Centricon 30,

MW 30000 cut-off; pre-washed by centrifuging with 2 mL water) while centrifiiging at 5000 g for 30-90 min, inject a 600 |JLL aliquot of the ultrafiltrate. (Prepare succinate buffer by dissolving 11.8 g succinic acid in 980 mL water, adjust pH to 4.0 with 10 M NaOH, make up to 1 L. Prepare the citrate/phosphate buffer by dissolving 12.9 g citric acid monohydrate, 10.9 g Na2HPO4, 37.2 g disodium EDTA dihydrate, and 29.2 g NaCl in 1 L water.) HPLCVARIABLES Column: 150 X 4.6 5 |xm PLRP-S (Polymer Labs) Mobile phase: Gradient. MeCN:MeOH: 10 mM oxalic acid 0:0:100 for 1 min, to 22:8:70 over 5 min, maintain at 22:8:70 for 11 min, return to initial conditions. Flow rate: 1 Injection volume: 600 Detector: UV 355 CHROMATOGRAM Retention time: 16.6 Limit of detection: 1.15 ng/mL Limit of quantitation: 2.22 ng/mL OTHER SUBSTANCES Extracted: chlortetracycline, demeclocycline, methacycline, minocycline, oxytetracycline, tetracycline Noninterfering: chloramphenicol, gentian violet, hydromycin B, ivermectin, spectinomycin, sulfa drugs KEYWORDS cow; SPE; ultrafiltrate REFERENCE
Carson, M.C. Simultaneous determination of multiple tetracycline residues in milk using metal chelate affinity chromatography J.AOAC Int., 1993, 76, 329-334

SAMPLE Matrix: tissue Sample preparation: Prepare an affinity column by filling a 10 mL column with 5 mL chelating Sepharose, allow to settle, wash with 20 mL 0.5% copper(II) sulfate solution, eliminate air bubbles by agitation, wash with 15 mL 50 mM pH 4 succinate buffer, do not allow to dry. Condition an Analytichem Bond Elut C18 SPE cartridge with 10 mL MeOH and 10 mL water, do not allow to dry. Homogenize 4 g minced kidney with 40 mL 50 mM pH 4 succinate buffer, sonicate for 10 min, centrifuge at 9000 rpm for 10 min, filter the supernatant through paper, repeat the extraction. Combine the supernatants and pass them through the affinity column at 5-7 mL/min, wash with 10 mL water, wash with 30 mL MeOH, wash with 20 mL water, elute with 50 mL 50 mM pH 4 succinate buffer containing 3.7% Titriplex III (ethylenedinitrilotetracacetic acid, disodium salt dihydrate). Add the eluate to the SPE cartridge at 5-7 mL/min, wash with 10 mL water, dry with air aspiration for 10 min, elute with 5 mL MeOHrMeCN 1:1, evaporate the eluate at 40 under a stream of nitrogen, dissolve the residue in 500 JJIL mobile phase, inject an aliquot. Protect from light through process. (The affinity columns may be re-used up to 15 times by washing with 20 mL water then 20 mL EtOH: water 20:80 then conditioning as described above.) HPLCVARIABLES Guard column: Perisorb RP-8 Column: two 300 X 100 5 |xm Chromspher C8 columns (cat. no. 28262) in series Mobile phase: MeCN: 10 mM pH 2 oxalic acid 20:80 Flow rate: 0.8 Detector: UV 365

CHROMATOGRAM Retention time: 26 Limit of quantitation: 30 ng/'g OTHER SUBSTANCES Simultaneous: ehlortetracycline, demethylchlortetracycline, methacycline, oxytetraeycline, tetracycline KEYWORDS kidney; SPE REFERENCE
Degroodt, J.M.; Wyhowski de Bukanski, B.; Srebrnik, S. Multiresidue analysis of tetracyclines in kidney by HPLC and photodiode array detection. J.Liq.Chromatogr., 1993, 16, 3515-3529

SAMPLE Matrix: tissue Sample preparation: Mince 0.1-0.3 g tissue with a scalpel and incubate at 37 with 0.5 mL water for 1 h. Add MeCN: 85% phosphoric acid: water 20:2:78 (muscle, renal medulla, lung) or MeOH: MeCN: 85% phosphoric acid .water 30:10:2:58 (renal cortex, liver) to a total volume of 1 mL, sonicate 30 min, filter (10 000 or 30 000 Da cutoff) by centrifuging at 2200 g for 30 min, inject 10-30 |xL aliquot of filtrate. HPLCVARIABLES Column: 220 X 2.1 Brownlee phenyl Spheri-5 MPLC cartridge Mobile phase: MeOH:MeCN:triethylamine:phosphoric acid:80 mM pH 2.4 sodium phosphate buffer 22.5:2.5:0.5:1.7:72.8 Column temperature: 60 Flow rate: 0.3-0.4 Injection volume: 10-30 Detector: UV 268; UV 345 CHROMATOGRAM Retention time: 6 Limit of detection: <5-10 ng/g KEYWORDS cow; muscle; renal cortex; renal medulla; liver; lung REFERENCE
Riond, J.L.; Hedeen, K.M.; Tyezkowska, K.; Riviere, J.E. Determination of doxycycline in bovine tissues and body fluids by high-performance liquid chromatography using photodiode array ultraviolet-visible detection. J.Pharm.ScL, 1989, 78, 44-47

ANNOTATED BIBLIOGRAPHY

Prevosto, J.M.; Beraud, B.; Cheminel, V.; Gaillard, Y; Mounier, C; Chaulet, J.F. Determination of doxycycline in human plasma and urine samples by high performance liquid chromatography. Application for drug monitoring in malaria chemoprophylaxis. Ann.Biol.Clin.(Paris), 1995, 53, 29-32 Colmenero, J.D.; Fernandez-Gallardo, L.C; Agiindez, J.A.G.; Sedeno, J.; Benitez, J.; Valverde, E. Possible implications of doxycycline-rifampin interaction for treatment of brucellosis. Antimicrob.Agents Chemother., 1994, 38, 2798-2802 [extracted rifampin; plasma; serum; papaverine (IS); LOQ 200 ng/mL] Hoogmartens, J.; Khan, N.H.; Vanderhaeghe, H.; Van der Leeden, A..L.; Oosterbaan, M.; Veld-Tulp, G.L.; Plugge, W.; Van der Vlies, C; Mialanne, D.; et al. A collaborative study of the analysis of doxycycline

hyclate by high-performance liquid chromatography on polystyrene-divinylbenzene packing materials. J.Pharm.Biomed.Anal, 1989, 7, 601-610 Nieder, M.; Jaeger, H. Selective quantification of doxycycline in human plasma and urine with optimized chromatography. Chromatographia, 1988, 25, 526-530 [column temp 30; plasma; urine; SPE; demeclocycline (IS); pharmacokinetics; non-interfering other tetracyclines, caffeine, nicotine, salicylic acid; LOQ 125 ng/mL] Dihuidi, K.; Kucharski, M.J.; Roets, E.; Hoogmartens, J.; Vanderhaeghe, H. Quantitative analysis of doxycycline and related substances by high-performance liquid chromatography. J.Chromatogr., 1985, 325, 413424 [column temp 60; bulk; tablets; capsules; simultaneous impurities, methacycline, oxytetracycline] Bocker, R. Rapid analysis of doxycycline from biological samples by high-performance liquid chromatography. J.Chromatogr., 1980, 187, 439-441 [whole blood; serum; tissue; mouse; liver] De Leenheer, A.P.; Nelis, H.J.C.F. Reversed-phase high-performance liquid chromatography of doxycycline. J.Chromatogr., 1977, 140, 293-299