Manidipine Hydrochloride Maprotiline

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MANIDIPINE HYDROCHLORIDE MAPROTILINE

CHEMISTRY CHEMISTRY

Manidipine is a dihydropyridine calcum-channel blocker. Maprotiline is a tetracyclic antidepressant. Its chem- Its chemical name is 2-[4-(diphenylmethyl)-1-piperazinyl] ical name is 3-(9,10-dihydro-9,10-ethanoanthracen-9- ethyl methyl (±)-1,4-dihydro-2,6-dimethyl-4-(m-nitro- yl)propyl(methyl)amine. Its molecular formula is C20H23N, phenyl)-3,5-pyridinedicarboxylate dihydrochloride. Its with a molecular weight of 277.4 and a CAS number of molecular formula is C35H38N4O6·2HCl, with a molecular 10262-69-8. Maprotiline hydrochloride occurs as a ﬁne weight of 683.6 and a CAS number of 126229-12-7. white to off-white, practically odorless, crystalline powder. Maprotiline hydrochloride is slightly soluble in water and freely soluble in chloroform and methanol, but practically insoluble in isooctane. METHOD

Assay 1 Jing et al. [1] reported the determination and METHODS pharmacokinetics of manidipine in human plasma by LC- MS. A Shimadzu liquid chromatograph equipped with a Assay 1 Volin [1] described a GC method for the model CTO20A column oven, model SIL20AC autoinjec- routine determination of tricyclic antidepressants in tor, and model DGU20A3 degasser was used. The station- human plasma with a specific nitrogen detector. A ary phase was an Elite (China) Hypersil ODS2 column Hewlett-Packard model HP5710A gas chromatograph was (200 × 4.6 mm, 5 ␮m particle size). The mobile phase con- equipped with a model 18765A nitrogen/phosphorus de- sisted of methanol and 5 mM ammonium acetate solution tector and model HP3390A data processor. The stationary × containing 0.1% acetic acid (85 : 15, vol/vol) and was de- phase was a Supelco coiled glass column (1.8 m 2mm livered isocratically at 0.7 mL/min. The injection volume i.d.) containing GP 3% SP2250 on 80/100-mesh Supelco- ␮ port. Temperatures for column, detector, and injector were was 10 L. ◦ ◦ ◦ ◦ A Shimadzu LCMS-2010EV mass spectrometer setat240C (or 250 C), 300 C, and 250 C, respectively. equipped with an electrospray ionization interface was The flow rate of nitrogen carrier gas was 40 mL/min. The ␮ operated in the positive-ion mode. The detector voltage injection volume was 2–5 L. was set at 1.75 kV. Quantification was performed in the Protriptyline at 1.0 mg/mL in methanol was used as selected-ion monitoring (SIM) mode using target ions at internal standard. A plasma sample or standard (3 mL) m/z 611.4 for manidipine and m/z 384 for felodipine. was mixed with 1.2 mL of saturated sodium carbonate Felodipine at 1 ␮g/mL in methanol was used as an (Na2CO3) by shaking vigorously for 2 min, extracted with internal standard. Standards were prepared by spiking 10 mL of n-hexane/isoamyl alcohol (97 : 3, vol/vol) con- ␮ blank human plasma with stock solutions of manidipine. taining 0.1 g/mL internal standard by shaking for 15 min, An aliquot of 1 mL of plasma sample or standard was mixed and centrifuged at 1000 g for 5 min. A portion (8 mL) with 50 ␮L of the internal standard, alkalinized by 0.1 mL of the organic phase was collected, mixed with 1.2 mL of 1% ammonia solution, vortexed, extracted with 4 mL of 0.9 M hydrochloric acid, shaken for 15 min, and cen- of n-hexane containing 2% isopropanol, and centrifuged. trifuged. The aqueous phase was collected, mixed vigor- Then 3 mL of organic layer was evaporated to dryness at ously with 500 mg of anhydrous sodium carbonate for 40◦C under reduced pressure, reconstituted in 100 ␮Lof 1 min, extracted with 3 mL of n-hexane/isoamyl alcohol 90% methanol in water, and assayed. Under these condi- without internal standard for 15 min, and centrifuged. tions, retention times of manidipine and felodipine were The organic phase was collected, mixed vigorously with 250 mg of anhydrous sodium sulfate, and centrifuged. The 5.8 and 5.6 min, respectively. ◦ Calibration curves for manidipine were constructed supernatant was collected, evaporated to dryness at 40 C ␮ over the range from 0.2 to 20 ng/mL. Correlation coeffi- under a stream of nitrogen, reconstituted in 120 Lof cients were 0.9996. Intraday and interday coefficients of methanol, and assayed. Relative retention times to inter- variation were less than 5.4% and 7.8%, respectively. The nal standard for amitriptyline, trimipramine, imipramine, limit of detection was 0.1 ng/mL. doxepin, nortriptyline, mianserin, iprindole, maprotiline, and clomipramine were 0.72, 0.74, 0.80, 0.83, 0.86, 0.89, 1.04, 1.11, and 1.22, respectively. A linear calibration curve for maprotilline was ob- REFERENCE tained in the concentration range of 25–175 ␮g/L. The recovery ranged from 86% to 102%. The coefficient of 1. Jing J. Ren W, Chen X, et al., Determination and pharma- variation was less than 8.6%. There was no interfer- cokinetics of manidipine in human plasma by HPLC/ESIMS, ence from perphenazine, nitrazepam, diazepam, levome- Biomed Chromatogr 21: 836–840 (2007). promazine, digoxin, atenolol, melperone, chlorpromazine,

Analytical Methods for Therapeutic Drug Monitoring and Toxicology, by Q. Alan Xu and Timothy L. Madden Copyright © 2011 Q. Alan Xu and Timothy L. Madden 299 P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

300 MAPROTILINE

thioridazine, lithium, flunitrazepam, fluphenazine, chlor- 10AVP UV detector was used. The stationary phase was a diazepoxide, propranolol, insulin, promazine, or lo- Macherey–Nagel Nucleosil 100-5-Protect 1 analytical col- razepam. umn (250 × 4.6 mm, 5 ␮m particle size). The mobile phase consisted of 25 mM monobasic potassium phosphate buffer Assay 2 Aymard et al. [2] reported an HPLC method for (pH 7.0) and acetonitrile (60 : 40) and was isocratically simultaneous quantification of imipramine, amitriptyline, delivered at 1 mL/min. UV detection was performed at maprotiline, fluoxetine, clomipramine, and their respective 230 nm. The injection volume was 100 ␮L. metabolites. A ThermoSeparation liquid chromatograph A stock solution of maprotiline at 1.0 mg/mL was pre- consisting of a model P1000 solvent delivery pump, model pared in methanol. Working solutions were prepared by AS3000 autosampler with a 100-␮Lloop,andaSpectra diluting this stock solution with water. Standards were Focus model photodiode array detector was used. The sta- prepared by spiking blank human serum with working so- tionary phase was a Waters Symmetry C18 column (250 × lutions. Melperone at 3000 ng/mL was used as an internal 4.6 mm, 5 ␮m particle size). The mobile phase consisted of standard. An aliquot of 1 mL of serum sample or standard 0.067 M monobasic potassium phosphate buffer (pH 3.0) was centrifuged at 13,000 g and at 4◦C for 10 min. An and acetonitrile (65 : 35, vol/vol) and was delivered iso- aliquot of 0.9 mL of the supernatant was separated; mixed cratically at 1.2 mL/min. UV detections were performed at with 0.1 mL of internal standard and 2.0 mL of 0.1 M 226, 254, and 400 nm. The total runtime of an injection monobasic potassium phosphate buffer (pH 6.0); loaded was 20 min. onto a Varian 3M-Empore extraction disk cartridge (3 mL) Stock solutions of drugs at 1.0 mg/mL each were pre- that was preconditioned with 1 mL of methanol followed pared in 0.01 M hydrochloric acid and stored at 4◦C. Stan- by 1 mL of water; pulled through the cartridge; washed dards were prepared by spiking drug-free human plasma sequentially with 1 mL of water, 1 mL of 1 M acetic acid, with stock solutions. Clovoxamine was used as an inter- 1mLofn-hexane, 2 mL of n-hexane–ethyl acetate (1 : 1), nal standard. An aliquot of 500 ␮L of a plasma sample or and 1 mL of methanol, eluted with 1 mL of 2-propanol–25% standard in a 15-mL Venoject silicone tube was alkalin- ammonium solution–dichloromethane (20 : 2: 78); evapo- ized with 250 ␮L of 2 M sodium carbonate, mixed with 100 rated to dryness, reconstituted with 250 ␮L of acetonitrile ␮Lof1␮g/mL internal standard, extracted with 10 mL and water (3 : 7); and assayed. Under these conditions, of n-hexane, shaken for 30 min, centrifuged at 3000 g for retention times for melperone and maprotiline were 8.8 10 min, and placed in a dry ice–acetone bath. The entire and 15.3 min, respectively. organic layer was collected, mixed with 200 ␮L of 0.03% Calibration curves for maprotiline were constructed phosphoric acid, shaken for 10 min, and centrifuged again. over the range from 10 to 500 ng/mL. Correlation coef- The acidic aqueous solution was collected and assayed. The ficients were 0.9997. Recovery of the drug from serum injection volume was 100 ␮L. Under these conditions, re- ranged from 95.4% to 97.8%. Intraassay and interassay tention times for clovoxamine, imipramine, maprotiline, coefficients of variation were less than 7.2% and 7.6%, re- amitriptyline, fluoxetine, and clomipramine were about spectively. There was no interference with the assay from 6.7, 9.8, 10.9, 11.5, 15.5, and 18.9 min, respectively. the following drugs and their metabolites (retention time A calibration curve for maprotiline was constructed in in minutes): sulpiride (4.1), O-desmethylvenlafaxine (4.8), the concentration range of 10–3000 ng/mL. The correla- 9-OH-risperidone (6.6), m-chlorophenylpiperazine (8.0), tion coefficient was greater than 0.998. The recovery of normirtazapine (8.3), zolpidem (10.2), nordoxepin (10.9), maprotiline from plasma was better than 78%. Within- diazepam (11.0), benperidol (11.5), normaprotiline (11.5), day and between-day coefficients of variation were 8.7% dibenzepine (11.5), opipramol (11.6), norfluoxetine (13.4), and 4.1%, respectively. The limit of quantification deter- norclozapine (14.4), haloperidol (15.3), norclomipramine mined at 226 nm was 5 ng/mL. There was no interference (19.2), trifluperidol (20.8), olanzapine (21.0), ziprasidone with this assay from the following drugs and their metabo- (26.4), promethazine (28.1), flupenazine (31.0), nefazodone lites (retention times in minutes): desmethylvenlafax- (32.5), chlorprothixene (36.4), thioridazine (43.2), pimozide ine (2.7), zopiclone (2.8), sulpiride (2.8), viloxazine (2.8), (44.1), carbamazepine, perazine, zotepine, valproate, zopi- zolpidem (3.3), venlafaxine (3.5), clozapine (4.5), chlor- clone, buspirone, lorazepam, and biperidene. diazepoxide (4.6), mianserine (5.7), doxepine (6.5), aminep- tine (6.5), loxapine (7.1), haloperidol (7.2), desipramine (8.9), desmethylmaprotiline (9.2), cyamemazine (9.4), car- REFERENCES bamazepine (9.5), fluvoxamine (10.0), nortriptyline (10.5), levomepromazine (11.5), trimipramine (13.1), norfluoxe- 1. Volin P, Therapeutic monitoring of tricyclic antidepressant tine (13.3), lorazepam (13.7), chlorpromazine (14.9), clon- drugs in plasma or serum by gas chromatography, Clin Chem 27: 1785–1787 (1981). azepam (15.4), desmethylclomipramine (16.9), clorazepate dipotassium (19.3), flunitrazepam (24.1), diazepam, and 2. Aymard G, Livi P, Pham YT, et al., Sensitive and rapid method for the simultaneous quantification of five antidepres- valproic acid. sants with their respective metabolites in plasma using high- performance liquid chromatography with diode-array detec- Assay 3 Frahnert et al. [3] reported the analysis of tion, J Chromatogr B 700: 183–189 (1997). maprotiline in human serum by HPLC for therapeutic 3. Frahnert C. Rao ML, Grasmader K, Analysis of eighteen an- drug monitoring. A liquid chromatograph consisting of a tidepressants, four atypical antipsychotics and active metabo- Bischoff 2200 pump, a Bischoff SDU2003 solvent degasser, lites in serum by liquid chromatography: a simple tool for ther- a Waters WISP 717 autosampler, and a Shimadzu SPD- apeutic drug monitoring, J Chromatogr B 794: 35–47 (2003). P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

MELITRACEN HYDROCHLORIDE 301

MEFRUSIDE ine, nitrazepam, oxazepam, oxprenolol, pindolol, prazosin, proparanolol, salbutamol, senokot, theophylline, and CHEMISTRY triﬂuoperazine.

Mefruside is a diuretic. Its chemical name is 4-chloro- REFERENCE N1-methyl-N1-(tetrahydro-2-methylfurfuryl)benzene-1,3- disulfonamide. Its molecular formula is C13H19ClN2O5S2, 1. Fullinfaw RO, Bury RW, Moulds RFW. Liquid chromatographic with a molecular weight of 382.9 and a CAS number of screening of diuretics in urine, JChromatogr415: 347–356 7195-27-9. (1987).

METHOD MELITRACEN HYDROCHLORIDE Assay 1 Fullinfaw et al. [1] described an HPLC CHEMISTRY method for screening chlorothiazide, hydrochlorothiazide, quinethazone, chlorthalidone, methyclothiazide, Melitracen is a tricyclic antidepressant. Its chem- clopamide, frusemide, metolazone, mefruside, bendroflu- ical name is 3-(9,10-dihydro-10,10-dimethyl-9-anthry- azide, cyclopenthiazide, and bumetanide in human urine. lidene)propyldimethylamine hydrochloride. Its molecular A Hewlett-Packard 1090A liquid chromatograph equipped formula is C H N·HCl, with a molecular weight of 327.9 with a photodiode-array detector was used. The stationary 21 25 and a CAS number of 10563-70-9. phase was a Merck LiChrosorb RP18 column (125 × 4 mm, 5 ␮m particle size). The column temperature was maintained at 50◦C. The mobile phase consisted of acetonitrile METHOD and 0.01 M phosphate buffer (pH 3.0) and was delivered at 1.5 mL/min in a gradient mode. The acetonitrile content of Assay 1 Kollroser and Schober [1] described the simul- mobile phase was increased from 10% at 1.5 min to 35% at taneous determination of seven tricyclic antidepressant 3.5 min. The total runtime was 10 min. UV detection was drugs—amitriptyline, nortriptyline, doxepin, dosulepin, performed at 271 nm. The injection volume was 5 ␮L. dibenzepin, opipramol, and melitracen—in human plasma Stock solutions of chlorothiazide, hydrochloroth- using HPLC-MS/MS with an ion-trap detector and direct iazide, quinethazone, chlorthalidone, methyclothiazide, injection. A TSP liquid chromatographic system consisting clopamide, frusemide, metolazone, mefruside, bendroflu- of two model P4000 quaternary pumps, a model AS3000 azide, cyclopenthiazide, and bumetanide at 1 mg/mL were autosampler, a vacuum degasser, and a six-port switch- separately prepared in ethanol and stored at 4◦C. Working ing valve was utilized. The stationary phase was a Waters solutions of these drugs were prepared by diluting stock Symmetry C18 column (150 × 3.0 mm, 5 ␮m particle size) solutions with ethanol. Standards were prepared by spik- protected by a Waters Symmetry C18 guard column (20 × ing blank human urine with working solutions. An aliquot 3.9 mm, 5 ␮m particle size). The mobile phase consisted of of 2 mL of urine samples was acidified with 2 mL of 1 M acetonitrile (A) and 0.1% formic acid in water (B) and was monobasic sodium phosphate buffer (pH 4.1), mixed with delivered at 0.6 mL/min in a step-gradient mode at 28% A 4 mL of ethyl acetate, vortexed for 2 min, and centrifuged for 4 min, increased to 70% A in 1 min, maintained at 70% at 1500 g for 5 min. The organic layer was separated, A for 3 min, returned to 28% A in 0.7 min, and kept at 28% mixed with 5 mL of 0.1 M dibasic sodium phosphate buffer A for another 3.3 min. (pH 7.5), vortexed for 2 min, and centrifuged. The organic A Finnigan LCQ ion-trap mass spectrometer equipped phase was collected, evaporated to dryness at 60◦C under with an APCI source was operated in the positive-ion nitrogen, reconstituted with 100 ␮L of 35% acetonitrile mode: vaporizer temperature 450◦C, capillary temperature in 0.01 M phosphate buffer (pH 3.0), and assayed. Under 200◦C, corona discharge intensity 5 ␮A, and sheath gas these conditions, retention times for chlorothiazide, hy- flow 60 units. Analytes were quantified using the follow- drochlorothiazide, quinethazone, chlorthalidone, methy- ing ion transitions: m/z 278.2 → 233.1 for amitriptyline, clothiazide, frusemide, metolazone, mefruside, bendroflu- m/z 264.2 → 233.1 for nortriptyline, m/z 296.2 → 251.2 azide, cyclopenthiazide, and bumetanide were about 1.9, for dibenzepin, m/z 280.2 → 235.1 for doxepin, m/z 296.1 2.2, 3.4, 4.2, 4.7, 5.1, 5.3, 6.2, 6.8, 7.2, and 9.3 min, re- → 225.1 for dosulepin, m/z 364.2 → 171.1 for opipramol, spectively (estimated from the published chromatogram). m/z 292.2 → 247.1 for melitracen, and m/z 419.1 →for Clopamide had the same retention time as did methycloth- lofepramine (internal standard). iazide. These two drugs were identified by comparing UV Stock solutions of all drugs at 1 mg/mL were separately spectra of the peak between 220 and 400 nm. prepared in methanol. Working solutions were prepared The mean recovery of mefruside from urine was 89%. with 0.1% formic acid in water. Lofepramine at 10 mg/L in There was no interference with this screening procedure 0.1% formic acid was used as an internal standard. Stan- from the following drugs: acetylsalicylic acid, allopuri- dards were prepared by spiking drug-free human plasma nol, alprenolol, atenolol, captopril, carbimazole, clonidine, with working solutions. Each plasma sample, control, or coloxyl, danthron, diazepam, digoxin, doxepin, gliben- standard was spiked with 10 ␮L of the internal standard clamide, hydralazine, indomethacin, labetalol, metformin, and diluted with an appropriate amount of 0.1% formic methyldopa, metoprolol, mianserin, minoxidil, nifedip- acid. An aliquot of 50 ␮L of this solution was injected onto a P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

302 MELOXICAM

Waters Oasis HLB online extraction column (50 × 2.1 mm, 2 ␮g/mL was used as an internal standard. An aliquot 30 ␮m particle size) with a mobile phase of 0.1% formic of 300 ␮L of plasma sample or standard was mixed with acid at a flow rate of 4 mL/min. The valve was directed 100 ␮L of internal standard solution and 100 ␮Lof1M to the waste from 0 to 0.3 min, automatically switched to hydrochloric acid, extracted with 3 mL of chloroform, vor- the analytical column for separation of drugs from 0.3 to texed for 10 min, and centrifuged at 3000 rpm for 10 min. 9.0 min, and then switched back to the waste. Under these The organic layer was separated, evaporated to dryness conditions, retention times of dibenzepin, opipramol, dox- at 50–55◦C, reconstituted with 30 ␮L of methanol and 20 epin, dosulepin, nortriptyline, amitriptyline, melitracen, mM monobasic sodium phosphate buffer (52 : 48, pH 7.6), and lofepramine were 2.06, 2.10, 2.40, 3.21, 3.59, 3.94, allowed to stand for 30 min, and assayed. Under these con- 5.82, and 7.36 min, respectively. ditions, retention times of meloxicam and carbamazepine Calibration curves for melitracen were constructed in were 5.3 and 6.1 min, respectively. the range from 10 to 800 ␮g/L. Correlation coefficients were Calibration curves for meloxicam were constructed over greater than 0.997. Intraassay and interassay accuracies the range from 0.02 to 2.0 mg/L. Correlation coefficients were within 93.2% and 109.1%, respectively. Intraassay were 0.9999. Recovery of the drug from plasma ranged and interassay coefficients of variation were less than 6.3% from 87.6% to 92.9%. Intraday and interday coefficients of and 8.2%. Limits of detection and quantification were 5 and variation were less than 1.8% and 5.8%, respectively. Limit 10 ␮g/mL, respectively. of detection was 0.01 mg/L.

Assay 2 Li et al. [2] reported the determination of meloxi- REFERENCE cam in human plasma by HPLC. A liquid chromatograph equipped with a XingDa model LP05 pump, a Shimadzu 1. Kollroser M, Schober C, Simultaneous determination of seven tricyclic antidepressant drugs in human plasma by direct- model SPD6A UV detector, a Waters manual injector, and injection HPLC-APCI-MS-MS with an ion trap detector, Ther a JiangShen model JS-3.0 chromatographic workstation Drug Monit 24: 537–544 (2002). was used. The stationary phase was a Kromasil ODS column (150 × 4.6 mm, 5 ␮m particle size) protected by a precolumn (30 × 4.6 mm, 10 ␮m particle size). The mobile phase consisted of methanol, water, acetonitrile, and MELOXICAM glacial acetic acid (600 : 500 : 50 : 20) containing 1.01 g of sodium heptanesulfonate and was isocratically delivered CHEMISTRY at 1.0 mL/min. UV detection was performed at 355 nm. The injection volume was 25 ␮L. Meloxicam is an oxicam derivative and an NSAID. A stock solution of meloxicam was prepared by dis- Its chemical name is 4-hydroxy-2-methyl-N-(5-methyl- solving 40 mg of meloxicam in 197.5 mL of 0.1 M sodium 2-thiazolyl)-2H-1,2-benzothiazine-3-carboxamide 1,1- hydroxide solution, sonicating, mixing with 100 mL of dioxide. Its molecular formula is C H N O S ,witha 14 13 3 4 2 water containing 3.4 g of monobasic potassium phosphate, molecular weight of 351.4 and a CAS number of 71125- and diluting to 500 mL with water. Working solutions 38-7. Meloxicam is a pale yellow powder. Meloxicam is were prepared by diluting this stock solution with water. practically insoluble in water, very slightly soluble in Standards were prepared by spiking blank human plasma ethanol and methanol, and slightly soluble in acetone. It with working solutions. An aliquot of 1 mL of plasma sam- is soluble in dimethylformamide. ple or standard was mixed with 4 mL of tetrahydrofuran, vortexed for 5 min, and centrifuged at 4000 rpm for 5 min. METHODS The supernatant was separated, evaporated to dryness at 80◦C, reconstituted with 1.0 mL of methanol, sonicated Assay 1 Huang et al. [1] described the analysis of meloxi- for 1 min, centrifuged at 4000 rpm for 5 min, and assayed. cam in human plasma. A Waters liquid chromatograph Under these conditions, the retention time of meloxicam equipped with a model 510 pump, model 486 variable- was 13 min. wavelength UV detector, and a Shimadzu model CR7Ae Calibration curves for meloxicam were constructed over integrator was used. The stationary phase was a Waters the range from 0.122 to 7.83 ␮g/mL. The average correla- Symmetry C18 column (150 × 3.9 mm, 5 ␮m particle tion coefficient was 0.9998. The recovery of the drug from size). The mobile phase consisted of methanol and 20 mM plasma ranged from 97.7% to 101.9%. Intraday and inter- monobasic sodium phosphate buffer (52 : 48), adjusted to day coefficients of variation were less than 6.8% and 9.6%, pH 5.2 with phosphoric acid, and was isocratically de- respectively. The limit of quantification was 0.122 ␮g/mL. livered at 1.2 mL/min. UV detection was performed at 271 nm. The injection volume was 20 ␮L. Assay 3 Sane et al. [3] reported an HPLC method for the A stock solution of meloxicam was prepared by dissolv- determination of meloxicam in human plasma. A Spec- ing 4 mg of meloxicam in 5 mL of 0.1 M sodium hydroxide tra Physics liquid chromatograph consisting of a model and diluting to 100 mL with water and stored at 4◦C. Work- SP8801 isocratic pump, model UV150 UV–visible detector, ing solutions were prepared by diluting this stock solution and a Rheodyne injector with a 50-␮L loop was used. The with water. Standards were prepared by spiking drug-free stationary phase was a Zorbax ODS column (250 × 4.6 mm, human plasma with working solutions. Carbamazepine at 5 ␮m particle size). The mobile phase consisted of 0.05 M P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

MELPERONE HYDROCHLORIDE 303

tri (ammonium citrate), methanol, and triethylamine (35 : umn (125 × 2 mm) with a LiChroCART 10-2 guard column 65 : 0.5) adjusted to pH 3.1 with phosphoric acid and was of the same packing material. Eluent A was 5 mM aqueous isocratically delivered at 1.0 mL/min. UV detection was ammonium formate adjusted to pH 3 with formic acid, and performed at 350 nm. eluent B was acetonitrile. The mobile phase was delivered Stock solutions of meloxicam at 500 ␮g/mL and piroxi- in a step-gradient mode: 40% B at 0.4 mL/min from 0 to cam at 1000 ␮g/mL were separately prepared in methanol. 5.5 min, 90% B at 0.7 mL/min from 5.5 to 8 min, 40% B at Working solutions were prepared by diluting stock solu- 0.65 mL/min from 8 to 9.5 min, and 40% B at 0.4 mL/min tions with mobile phase. Standards were prepared by for- from9.5to10min. tifying blank human plasma with working solutions. An An Agilent Technologies AT1100 atmospheric-pressure aliquot of 1 mL of plasma or standard was mixed with chemical ionization electrospray (APCI) LC/MSD system 0.2 mL of 5% perchloric acid, vortexed for 1 min, extracted was used with the following parameters: drying gas, nitro- with 10 mL of methylene chloride, shaken for 10 min, and gen (7 L/min, 300◦C); nebulizer gas, nitrogen (172.5 kPa); centrifuged at 1500 rpm for 10 min. An aliquot of 8 mL capillary voltage, 4000 V; vaporizer temperature, 400◦C; of the organic layer was separated, evaporated to dryness corona current, 5.0 ␮A; fragmenter voltage, 100 V; and at 40◦C under a stream of nitrogen, reconstituted in mo- positive selected-ion monitoring (SIM) mode. The full scan bile phase, and assayed. Under these conditions, retention in the 100- and 200-V traces was made with the follow- times of meloxicam and piroxicam were 6.1 and 4.1 min, ing ions (m/z): 342, 313, 370, 427, 411, 327, 380, 264, 343, respectively. 340, 376, 420, 401, 298(IS), 438, 332, 435, and 462. Melper- Calibration curves for meloxicam were constructed over one was quantitated in the SIM mode at 100 V fragment the range from 0.10 to 3.00 ␮g/mL. Correlation coeffi- voltage at m/z 264 in a time window of 0–3.8 min. cients were 0.999. The recovery of meloxicam from plasma Trimipramine-d3 0.01 mg/mL in methanol was used ranged from 97.0% to 102.4%. Intraday and interday coef- as the internal standard. A stock solution of melperone ficients of variation were less than 5.7% and 6.4%, respec- 1 mg/mL was prepared in methanol. Working solutions tively. Limit of detection and quantification were 0.05 and were prepared by diluting the stock solution. Standards 0.10 ␮g/mL, respectively. were prepared by spiking pooled blank human plasma with working solutions. A plasma sample or standard (0.5 mL) was diluted with 2 mL of purified water; mixed with 0.05 REFERENCES mL of the internal standard for 15 s; loaded onto a Sepa- rtis Isolute Confirm HCX solid-phase cartridge that was 1. Huang Y, Liang M-Z, Yu Q, et al., RP-HPLC determination of meloxicam in human plasma, Yaowu Fenxi Zazhi 22: 183–185 preconditioned with 1 mL of methanol followed with 1 (2002). mL of water; washed sequentially with 1 mL of puri- 2. Li Y, Wang G-F, Wu Y-F, HPLC determination of meloxicam in fied water, 1 mL of 0.01 M hydrochloric acid, and 2 mL human plasma, Yaowu Fenxi Zazhi 21: 33–36 (2000). of methanol; dried under vacuum; eluted with 1 mL of methanol–aqueous ammonia (98 : 2, vol/vol); evaporated 3. Sane RT, Surve V, Francis M, Reversed phase high perfor- ◦ mance liquid chromatographic determination of meloxicam to dryness at 56 C under a gentle stream of nitrogen; re- from human plasma using an internal standard, India Drugs constituted with 50 ␮L of methanol; and assayed. The in- 37: 251–254 (2000). jection volume was 2 ␮L. The least-squares (1/C2) calibration curve for melperone was calculated by plotting the peak area ratios of the MELPERONE HYDROCHLORIDE target ion of the drug against that of the internal standard in the range of 0.02–0.5 mg/L. The correlation coefficient was 0.998. Within-day and between-day coefficients CHEMISTRY of variation were 2.7% and 6.9%, respectively. Accuracy in the relative percentage error was less than 6.0%. The re- Melperone hydrochloride is an antipsychotic agent. covery ranged from 35.5% to 45.1%. The limits of detection Its chemical name is 4-fluoro-4-(4-methylpiperidino) and quantification were 0.005 and 0.02 mg/L, respectively. butyrophenone hydrochloride. Its molecular formula is This assay has been validated for simultaneous determina- C H FNO·HCl, with a molecular weight of 299.8 and a 16 22 tion of amisulpride, bromperidol, clozapine, droperidol, flu- CAS number of 1622-79-3. penthixol, fluphenazine, haloperidol, melperone, olanzapine, perazine, pimozide, risperidone, sulpiride, zotepine, METHOD zuclopenthixol, norclozapine, clozapine N-oxide, and 9- hydroxyrisperidone. Assay 1 Kratzsch et al. [1] reported a validated assay for the simultaneous determination of 15 neuroleptics and REFERENCE three of their metabolites in plasma, including melperone by liquid chromatography/mass spectrometry with atmo- 1. Kratzsch C, Peters FT, Kraemer T, et al., Screening, library- spheric pressure chemical ionization. An Agilent Technolo- assisted identification and validated quantification of fifteen gies AT1100 Series HPLC system consisted of a binary neuroleptics and three of their metabolites in plasma by liquid pump, autosampler, and degasser. The stationary phase chromatography/mass spectrometry with atmospheric pres- was a Merch LiChroCART Superspher 60 RP Select B col- sure chemical ionization, J Mass Spectrom 38: 283–295 (2003). P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

304 MERCAPTOPURINE

MEPINDOLOL SULFATE MERCAPTOPURINE

CHEMISTRY CHEMISTRY

Mepindolol is a noncardioselective ␤-blocker. Its chem- Mercaptopurine is an antineoplastic. Its chemical name ical name is 1-isopropylamino-3-(2-methylindol-4-yloxy) is 6-mercaptopurine monohydrate. Its molecular formula propan-2-ol sulfate. Its molecular formula is (C15H22 is C5H4N4S·H2O, with a molecular weight of 170.2 and N2O2)2.H2SO4, with a molecular weight of 622.8 and a CAS a CAS number of 6112-76-1. Mercaptopurine occurs as a number of 56396-94-2. yellow, odorless or practically odorless, crystalline powder. Mercaptopurine is insoluble in water, acetone, and ether. It is soluble in hot alcohol and in dilute alkali solutions.

METHOD METHODS Assay 1 Millerioux et al. [1] described the determination of mepindolol in plasma and urine using HPLC Assay 1 Yuan et al. [1] reported the simultaneous deter- with electrochemical detection. A Waters liquid chromato- mination of azathioprine and 6-mercaptopurine in human graph equipped with a model M590 pump, model 710B plasma by HPLC. A Waters LC system consisting of model WISP autosampler, an ESA model 5100A Coulochem elec- 510 pumps, model 490E UV detector, model U6K injec- trochemical detector with a model 5020 guard cell and tor, and model P5200 integrator was used. The stationary model 5011 analytical cell, and a Houston Omniscribe phase was a Spherisorb C18 stainless-steel column (10 ␮m recorder was used. The stationary phase was a Waters particle size). The mobile phase consisted of methanol, wa- ␮Bondapak C18 column (10 ␮m particle size). The mo- ter, and diethylamine (200 : 800 : 10, vol/vol/vol) contain- bile phase consisted of acetonitrile, PIC-B7, acetic acid, ing 1 g/L sodium acetate, adjusted to pH 8.5 with glacial andwater(300:20:3:677, vol/vol/vol/vol) and was iso- acetic acid. The flow rate was 0.6 mL/min. UV detection cratically delivered at 1 mL/min. The electrochemical de- was performed at 313 nm and 0.05 AUFS. tector was operated at 0.88 V for the guard cell, 0.40 V A stock solution of azathioprine at 500 ␮g/mL was pre- for the first electrode, and 0.68 V for the working elec- pared in 0.05 M sodium hydroxide solution. A stock so- trode of the analytical cell. The injection volume was lution of 6-mercaptopurine at 100 ␮g/mL was prepared by 20–100 ␮L. dissolving 10 mg of the reference standard in 5 mL of water Stock solutions of mepindolol and pindolol at 500 ␮g/mL and 0.2 mL of 1 M sodium hydroxide solution and filling were separately prepared in methanol. Working solutions to the 100 mL mark with sodium acetate buffer (pH 6). were prepared by diluting stock solutions with methanol. Standards were prepared by spiking blank human plasma Standards in plasma or in urine were prepared by spiking with stock solutions. An aliquot of 0.5 mL of plasma or blank plasma or blank urine with working solutions. Pin- standard was spiked with 15 ␮L of metronidazole (inter- dolol at 1000 ng/mL in methanol was used as an internal nal standard) at 40 ␮g/mL in water, mixed with 0.4 mL standard. An aliquot of 1 mL of plasma sample or stan- of 10% trichloroacetic acid, vortexed, and centrifuged at dard was mixed with 100 ␮L of methanol, 100 ␮Lofthe 4000 rpm for 10 min. The supernatant was separated internal standard solution, 2 mL of titrisol buffer (pH 9), and assayed. Under these conditions, retention times for and 10 mL of methylene chloride; shaken for 10 min, and 6-mercaptopurine, metronidazole, and azathioprine were centrifuged at 3800 rpm for 10 min. An aliquot of 8 mL about 5.6, 7.0, and 12 min, respectively (estimated from of the organic layer was separated, mixed with 250 ␮L the published chromatogram). of 0.1 M acetic acid, shaken for 10 min, and centrifuged Calibration curves for 6-mercaptopurine were con- at 3800 rpm for 10 min. The upper aqueous layer was structed over the range from 0.5 to 4 ␮g/mL. Correla- separated, and assayed. Under these conditions, retention tion coefficients were 0.9994. The average recovery of 6- times of pindolol and mepindolol were 7.5 and 9.5 min, mercaptopurine from plasma was 103.4%. Intraday and respectively. interday coefficients of variation were less than 2.6% and Calibration curves for mepindolol were constructed over 10.0%, respectively. Cyclosporine A, prednisone, methyl- the range from 0 to 100 ng/mL in plasma and from 0 prednisolone, and 4-oxadocosane-1,2-diol did not interfere to 800 ng/mL in urine. The recovery of mepindolol from with this method. plasma was 75%. The coefficient of variation of the assay was 5.7%. The limit of detection was 0.625 ng/mL. Assay 2 Wusk et al. [2] reported the therapeutic drug monitoring of thiopurine drugs in patients using HPLC. A Varian HPLC system consisting of a model 9010 pump, model 9100 autosampler, and model 9050 UV–visible de- REFERENCE tector was used. The stationary phase was a Phenomenex × ␮ 1. Millerioux L, Ntzanis L, Julian B, et al., Highly sensitive Aqua C18 column (250 4.6 mm, 5 m particle size). measurement of mepindolol in biological fluids using HPLC Solvent A was a mixture of 5 mM ammonium acetate and electrochemical detection, Chromatographia 24: 377–379 buffer (pH 3), methanol, and acetonitrile (95 : 2.5 : (1987). 2.5, vol/vol/vol) containing 0.2% hexanesulfonic acid and P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

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solvent B was a mixture of 5 mM ammonium acetate ceftazidime, cefuroxime, meropenem, and piperacillin—in buffer (pH 3), methanol, and acetonitrile (5 : 75 : 20, human plasma by HPLC-UV. A Waters Alliance 2695 Sep- vol/vol/vol). The mobile phase was delivered at 1.0 mL/min aration module consisting of a quaternary pump, degasser, in a gradient mode. Solvent A was delivered at 100% from autosampler, and a Waters 2996 photodiode-array detector 0 to 5 min, decreased to 70% from 5 to 10 min, and held was used. The stationary phase was a Waters Symmetry at 70% from 10 to 20 min. UV detection was performed at C8 analytical column (250 × 4.6 mm, 5 ␮m particle size) 325 nm. protected by a guard column (20 × 4.6 mm) of the same Stock solutions of 6-mercaptopurine, 6-thioguanine, packing material. The mobile phase consisted of acetoni- and 6-methylmercaptopurine at 1 mg/mL were separately trile (A) and phosphate buffer (B) that was prepared by prepared in 0.01 M sodium hydroxide and stored at mixing 197 mL of 0.067 M monobasic potassium phosphate −20◦C. Working solutions were prepared by diluting stock solution with 803 mL of 0.067 M dibasic sodium phosphate solutions. Standards were prepared by spiking blank solution and adjusting to pH 7.4. It was delivered at 5% human whole blood with working solutions. An aliquot of A for 5 min, linearly increased to 50% A in 20 min, held 500 ␮L of sample or standard was mixed with 75 ␮Lof at 50% A for 1 min, and returned to the initial condition ice-cold perchloric acid, vortexed for 3 min, placed on ice in 5 min. The flow rate was 1 mL/min. UV detection was for 10 min, heated at 100◦C for 60 min, cooled to room performed at 256 nm for cefepime and ceftazidime, 270 nm temperature, mixed with 150 ␮L of 4 M dibasic potassium for cefuroxime and ceforanide, 300 nm for meropenem, and phosphate buffer, vortexed, placed on ice for 10 min, 220 for piperacillin. The injection volume was 30 ␮L. and centrifuged at 10,000 g for 5 min. The supernatant Stock solutions of cefepime, ceftazidime, cefuroxime, was separated and assayed. Under these conditions, meropenem, and piperacillin at 2.0 mg/mL each were retention times for 6-mercaptopurine, 6-thioguanine, and prepared in water. Standards were prepared by spiking 6-methylmercaptopurine were 9.2, 11.5, and 15.8 min, blank human plasma with stock solutions. Ceforanide at respectively. 125 ␮g/mL in water was used as an internal standard. An Calibration curves for mercaptopurine were con- aliquot of 0.5 mL of plasma or standard was mixed with structed over the range from 25 to 2000 ng. This method 50 ␮L of the internal standard solution, loaded onto a Wa- was used to analyze whole-blood samples from 200 ters C18 Sep-Pak solid-phase extraction cartridge that was patients. preconditioned with 3 × 1 mL of methanol followed by 2 × 1 mL of phosphate buffer, drawn through the cartridge, washed 4 × 300 ␮L of phosphate buffer and 2 × 300 ␮Lof REFERENCES 5% methanol in phosphate buffer, eluted with 3 × 500 ␮Lof methanol, evaporated to dryness at 30◦C under a stream 1. Yuan C, Sun C, Cao X, et al., Simultaneous determination ␮ of azathioprine and 6-mercaptopurine in human plasma by of nitrogen, reconstituted with 100 L of the initial mo- HPLC, Chinese J Hosp Pharm 14: 485–487 (1994). bile phase, and assayed. Under these conditions, retention 2. Wusk B, Kullak-Ublick GA, Rammert C, et al., Therapeutic times for ceftazidime, cefepime, ceforanide, meropenem, drug monitoring of thiopurine drugs in patients with inflam- cefuroxime, and piperacillin were 11.50, 13.00, 16.80, matory bowel disease or autoimmune hepatitis, Eur J Gas- 17.80, 19.95, and 22.36 min, respectively. troenterol Hepatol 16: 1407–1413 (2004). Calibration curves for meropenem were constructed over the range from 2.5 to 60 ␮g/mL. Correlation coefficients were greater than 0.994. The accuracy was better than 94.3%. Recovery of the drug from plasma ranged MEROPENEM from 74.3% to 79.7%. Intraday and interday coefficients of variation were 3.1% and 5.9%, respectively. The limit of CHEMISTRY quantification was 0.5 ␮g/mL. Meropenem is a carbapenem ␤-lactam antibacte- Assay 2 Ozkan et al. [2] reported an HPLC method for the rial. Its chemical name is (4R,5S,6S)-3-[(3S,5S)-5- determination of meropenem in human serum and urine. dimethylcarbamoylpyrrolidin-3-ylthio]-6-[(R)-1-hydroxy- A Waters liquid chromatograph consisting of a model ethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-ca- 510 pump, mode 481 UV detector, and model 717 Plus rboxylic acid trihydrate. Its molecular formula is autosampler was used. The stationary phase was a Waters C H N O S·3H O, with a molecular weight of 437.5 17 25 3 5 2 LC column (150 × 4.6 mm, 5 ␮m particle size). The mobile and a CAS number of 119478-56-7. Meropenem occurs 18 phase consisted of 15 mM monobasic potassium phosphate as colorless to white crystals. Meropenem is sparingly buffer, acetonitrile, and methanol (84 : 12 : 4, vol/vol/vol), soluble in water, very slightly soluble in ethanol, and adjusted to pH 2.8 with phosphoric acid, and was isocrati- practically insoluble in acetone and ether. It is soluble in cally delivered at 1.0 mL/min. UV detection was performed dimethylformamide. at 307.6 nm. The injection volume was 50 ␮L. Stock solutions of meropenem and cefepime at 1.0 METHODS mg/mL were separately prepared in distilled water. Ce- fepime was used as an internal standard. Standards in Assay 1 Denooz and Charlier [1] reported the simultane- serum or urine were prepared by spiking blank human ous determination of five ß-lactam antibiotics—cefepime, serum or urine with stock solutions. For serum, an aliquot P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

306 MESALAZINE

of 500 ␮L of serum sample or standard was mixed with a a 20-␮L loop, and an ESA Coulochem model 5200A elec- desired amount of the internal standard solution, vortexed trochemical detector with a model 5021 conditioning cell for 30 s, centrifuged at 4000 g for 10 min, and assayed. For and a model 5011 analytical cell was utilized. The condi- urine, an aliquot of 2 mL of sample or standard was mixed tioning cell placed between the column and the analytical with a desired amount of the internal standard solution, cell was used to minimize the background noise of mobile vortexed for 30 s, loaded onto a Sep-Pak NH2 solid-phase phase and was set at −350 mV. The analytical cell was set extraction cartridge, washed with 2 mL of distilled water, at −50 mV for the first electrode (E1) and at + 450 mV for eluted with 1 mL of 0.1 N hydrochloric acid, and assayed. the second electrode (E2). The stationary phase was a Kro- Under these conditions, retention times of cefepime and masil KR100 5C18 analytical column (150 × 4.6 mm, 5 ␮m meropenem were 6.0 and 7.5 min, respectively. particle size) protected by a disposable Supelco Pelliguard Calibration curves for meropenem were constructed precolumn (20 × 4.6 mm, 40 ␮m particle size). The mo- over the range from 500 to 10,000 ng/mL in serum. Corre- bile phase consisted of 10 mM dibasic sodium phosphate lation coefficients were 0.999. The recovery of meropenem buffer and methanol (90 : 10, vol/vol), adjusted to pH 3.0 from serum was 98.1%. The coefficient of variation of the with 100 mM sodium hydroxide solution. The phosphate assay was 1.2%. Limit of detection was 108.4 ng/mL. buffer contained 0.1 mM EDTA, 100 mM citric acid, and Calibration curves for meropenem were also con- 2 mM heptanesulfonic acid. The flow rate was 0.8 mL/min. structed over the range from 750 to 10,000 ng/mL in The injection volume was 25 ␮L. urine. Correlation coefficients were 0.998. The recovery Stock solutions of 5-aminosalicylic acid, acetyl-5- of meropenem from urine was 96.1%. The coefficient of aminosalicylic acid (metabolite), and N-propionyl-4- variation of the assay was 1.6%. Limit of detection was aminosalicylic acid (internal standard) at 0.5 mg/mL were 179.3 ng/mL. separately prepared in methanol. Standards were prepared by diluting stock solutions with drug-free human plasma and were stored at −20◦C. An aliquot of 500 ␮L REFERENCES of plasma sample or standard was mixed with 100 ␮L of internal standard and 1.0 mL of methanol, vortexed 1. Denooz R, Charlier C, Simultaneous determination of five for 1 min, and centrifuged at 1500 g for 10 min. The su- ß-lactam antibiotics (cefepim, ceftazidim, cefuroxim, pernatant was collected and assayed. Under these condi- meropenem and piperacillin) in human plasma by high- performance liquid chromatography with ultraviolet detection, tions, retention times for 5-aminosalicylic acid, acetyl-5- J Chromatogr B 864: 161–167 (2008). aminosalicylic acid, and N-propionyl-4-aminosalicylic acid 2. Ozkan Y, Kucukguzel I, Ozkan SA, et al., A rapid, sensitive were4.9,9.9,and12.4min,respectively. high performance liquid chromatographic method for the de- Calibration curves were constructed over the range termination of meropenem in pharmaceutical dosage form, hu- from 50 to 3200 ng/mL for both 5-aminosalicylic acid man serum and urine, Biomed Chromatogr 15: 263–266 (2001). and acetyl-5-aminosalicylic acid. Correlation coefficients were greater than 0.9996 for 5-aminosalicylic acid and 0.9993 for acetyl-5-aminosalicylic acid. The recoveries of 5-aminosalicyclic acid, acetyl-5-aminosalicylic acid, and MESALAZINE N-propionyl-4-aminosalicylic acid in human plasma were 93%, 88%, and 95%, respectively. Intraday coefficients of variation were less than 1.6% for 5-aminosalicylic acid and CHEMISTRY 2.2% for its metabolite. Interday coefficients of variation Mesalazine is an anti-inflammtory drug. Its chemical were less than 2.9% for both the drug and its metabo- name is 5-amino-2-salicylic acid. Other names include 5- lite. The limits of detection were 5 ng/mL for the drug and aminosalicylic acid, 5-ASA, Asacol, Pentasa, and Rowasa. 10 ng/mL for its metabolite. The limit of quantification was 50 ng/mL for both compounds. Its molecular formula is C7H7NO3, with a molecular weight of 153.1 and a CAS number of 89-57-6. Mesalazine occurs as light tan to pink needle-shaped crystals, odorless Assay 2 Nobilis et al. [2] reported the determination of 5- or with a slight characteristic odor. Mesalazine is slightly aminosalicylic acid and its metabolites in human plasma soluble in water; very slightly soluble in dehydrated al- using HPLC with UV detection and fluorescence detection cohol, acetone, and methanol; and practically insoluble in after derivatization. A ThermoElectron chromatograph butanol, chloroform, dichloromethane, ether, ethyl acetate, consisting of a model P4000 quaternary gradient pump, ␮ n-hexane, and propanol. It is soluble in dilute hydrochloric model AS3000 autosampler with a 100- L loop, model acid and dilute alkali hydroxides. UV6000 LP photodiode-array detector, model FL3000 fluorescence detector, model SN4000 system controller, and a model SCM1000 solvent degasser was used. The station- METHODS ary phase was a Merck LiChroCART Purospher RP18e column (250 × 4 mm, 5 ␮m particle size) protected by a Assay 1 Palumbo et al. [1] reported the simultaneous LiChroCART precolumn (4 × 4 mm) of the same packing analysis of 5-aminosalicylic acid and its metabolite in hu- material. The mobile phase consisted of acetonitrile and man plasma using HPLC with electrochemical detection. 0.01 M dibasic sodium phosphate buffer, pH 3 (15 : 85, A liquid chromatographic system consisting of a Waters vol/vol) and was delivered at 1 mL/min. UV detection was model 515 pump, a Rheodyne model 7725i injector with performed at 313 nm, and the fluorescence detector was P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

MESUXIMIDE 307

set at an excitation wavelength of 300 nm and an emission voltage at 20 V. Analytes were monitored in multiple- wavelength of 406 nm. The injection volume was 100 ␮L. reaction monitoring (MRM) mode at the following ion tran- The total runtime was 23 min. sitions: m/z 152 → 108 for 5-ASA and 4-ASA, m/z 194 → Stock solutions of all aminosalicylic compounds under 107 for N-acetyl-5-ASA, and m/z 194 → 108 for N-acetyl- study at 1 × 10−3 M were prepared by dissolving equimo- 4-ASA. lar amounts of sodium hydroxide in water. A stock solu- Stock solutions of 5-ASA, N-acetyl-5-ASA, 4-ASA, and tion containing 1 × 10−4 M N-acetyl-4-aminosalicylic acid N-acetyl-4-ASA at 100 ␮g/mL were separately prepared and 1 × 10−3 M 4-aminosalicylic acid in water was used in methanol and stored at −20◦C. The internal standard as an internal standard. An aliquot of 1 mL of plasma solution was prepared by diluting stock solutions of 4-ASA was spiked with 30 ␮L of internal standard, shaken vig- and N-acetyl-4-ASA. Standards were prepared by spik- orously, mixed with 20 ␮L of concentrated perchloric acid, ing blank human plasma with stock solutions of 5-ASA vortexed for 1 min, and centrifuged at 2000 g for 12 min. and N-acetyl-5-ASA. An aliquot of 490 ␮Lofplasmaor The supernatant was collected, mixed with 600 ␮Lof standard was spiked with 10 ␮L of the internal standard, phosphate buffer that was prepared by mixing 800 mL mixed with 1.0 mL of methanol, stirred, and centrifuged of 0.067 M dibasic sodium phosphate and 200 mL of 0.067 at 12000 g for 10 min. An aliquot of 1.2 mL of the su- M monobasic potassium phosphate, mixed with 20 ␮Lof pernatant was collected, evaporated to dryness under vac- propionic anhydride, shaken vigorously, allowed to stand uum, reconstituted with 500 ␮Lof50mMaceticacidaque- at 25◦C for 20 min, mixed with 50 ␮L of concentrated hy- ous solution, centrifuged, and assayed. Under these con- drochloric acid, extracted with 3 mL of ethyl acetate, cen- ditions, retention times of 5-ASA, N-acetyl-5-ASA, 4-ASA, trifuged at 2000 g for 12 min, and stored at −34◦Cfor60 and N-acetyl-4-ASA were about 2.1, 4.5, 6.1, and 6.4 min, min. The organic phase was collected, evaporated at 45◦C respectively. under a stream of nitrogen, reconstituted with 600 ␮L Calibration curves were constructed in the range from of the mobile phase, and assayed. 5-Aminosalicylic acid 50 to 4000 ng/mL for both 5-ASA and N-acetyl-5-ASA. Cor- (5-ASA) was derivatized to N-propionyl-5-aminosalicylic relation coefficients were greater than 0.997. The average acid. Under these conditions, retention times of 5-ASA, recoveries were 90.8% and 95.2% for 5-ASA and N-acetyl- N-formyl-5-ASA, N-acetyl-5-ASA, 4-ASA, N-acetyl-4-ASA, 5-ASA, respectively. Within-batch and between-batch co- N-propionyl-5-ASA, N-propionyl-4-ASA, and N-butyryl-5- efficients of variation were less than 6.3% and 11% for ASA were about 2.8, 4.6, 5.2, 6.8, 7.5, 9.2, 16.1, and 5-ASA and 8.0% and 10% for N-acetyl-5-ASA. Limits of de- 18.8 min, respectively. tection and quantification were 15 and 50 ng/mL for both Calibration curves for N-propionyl-5-ASA (the deriva- compounds. tive of 5-ASA) were constructed over the range from 318 to 4967 pmol/mL. Correlation coefficients were greater than 0.9997. The recovery was 81%. Intraday and interday co- REFERENCES efficients of variation were less than 9.6% and 23.8%, respectively. Limit of detection and quantification were 50 1. Palumbo G, Bacchi S, Primavera L, et al., A validated HPLC and 318 pmol/mL, respectively. method with electrochemical detection for simultaneous assay Calibration curves for N-acetyl-5-ASA (the principal of 5-aminosalicylic acid and its metabolite in human plasma, metabolite of 5-ASA) were constructed over the range from Biomed Chromatogr 19: 350–354 (2005). 126 to 4967 pmol/mL. Correlation coefficients were greater 2. Nobilis M, Vyb´ıralovaZ,Sl´ adkov´ a´ K, et al., High-performance than 0.9993. The recovery was 78%. Intraday and interday liquid chromatographic determination of 5-aminosalicylic acid coefficients of variation were less than 6.2% and 13.6%, re- and its metabolites in blood plasma, JChromatogrA1119: spectively. Limit of detection and limit of quantification 299–308 (2006). were 20 and 126 pmol/mL, respectively. 3. Pastorini E, Locatelli M, Simoni P, et al., Development and validationof a HPLC-ESI-MS/MS method for the determina- Assay 3 Pastorini et al. [3] developed an LC-MS/MS tion of 5-aminosalicylic acid and its major metabolite N-acetyl- method for the determination of 5-aminosalicylic acid (5- 5-aminosalicylic acid in human plasma, J Chromatogr B 872: ASA) and its major metabolite , N-acetyl-5-aminosalicylic 99–106 (2008). acid, in human plasma. A Waters model 2695 Alliance system was used. The stationary phase was a Phenomenex Synergi Hydro-RP column (150 × 2.0 mm, 4 ␮m particle size) protected by a guard column (10 × 2.0 mm, 4 ␮m par- MESUXIMIDE ticle size). Solvent A was 17.5 mM acetic acid in water and solvent B, acetonitrile. The mobile phase was delivered at CHEMISTRY 0.2 mL/min at 15% B for 8 min, then at 80% B for 5 min, and at 15% B again for 12 min. The injection volume was Mesuximide is a succinimide antiepileptic. Its chemical 4 ␮L. name is N,2-dimethyl-2-phenylsuccinimide. Its molecular A Micromass Quattro triple quadrupole mass spectrom- formula is C12H13NO2, with a molecular weight of 203.2 eter was operated in negative-ion mode: nebulizer gas (ni- and a CAS number of 77-41-8. Mesuximide occurs as a trogen) at 100 L/h, desolvation gas (nitrogen) at 675 L/h, white to grayish-white crystalline powder. Mesuximide is ion-source block temperature at 120◦C, desolvation tem- soluble 1 in 350 of water, 1 in 3 of alcohol, 1 in < 1of perature at 230◦C, capillary voltage at 2.5 kV, and cone chloroform, and 1 in 2 of ether. P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

308 METFORMIN HYDROCHORIDE

METHOD METHODS

Assay 1 Varma [1] reported the simultaneous determi- Assay 1 Jiang et al. [1] reported an HPLC method for nation of mesuximide, ethouximide, phenobarbital, carba- the determination of metformin in human plasma. A Shi- mazepine, primidone, and phenytoin in plasma. A Varian madzu LC2010C liquid chromatographic sytem was used. series 2000 Aerograph equipped with a flame ionization The stationary phase was a GL Science Inertsil ODS3 sil- detector and a Hewlett-Packard model 3380A integrator ica column (150 × 4.6 mm, 5 ␮m particle size) protected by was used. The stationary phase was a glass column (6 ft a Dikma precolumn. The column temperature was main- × 1 ◦ 8 in.) packed with 3% QF-1 on Gas-Chrom Q (100/120 tained at 35 C. The mobile phase consisted of methanol mesh). The injector and detector temperatures were set at and 5 mM dibasic sodium phosphate buffer (pH 6.6) 230◦C and 280◦C, respectively. The oven temperature was (56 : 44) containing 0.35 mM sodium dodecylsulfate and setat115◦C for 2 min and then programmed to 175◦Cat was isocratically delivered at 1.2 mL/min. UV detection 4◦C/min. The carrier gas was nitrogen at 20 mL/min. The was performed at 234 nm. The injection volume was 20 ␮L. injection volume was 1–2 ␮L. Standards were prepared by spiking blank human A stock solution of these six drugs at 1 mg/mL plasma with a known quantity of metform. Pirenzepine each was prepared in methanol. 5-(p-Methylphenyl)-5- at 200 mg/L was used as an internal standard. An aliquot phenylhydantoin at 1 mg/mL in methanol was used as of 0.5 mL of plasma sample or standard was spiked with an internal standard. Standards were prepared by spik- 10 ␮L of the internal standard solution, mixed with 50 ␮L ing blank plasma with the stock solution. An aliquot of of 1 M hydrochloric acid, extracted with 1.5 mL of acetoni- 1 mL of plasma or standard was spiked with 20 ␮Lof trile, vortexed for 1 min, and centrifuged at 4000 rpm for 6 the internal standard solution, buffered with 1 mL of 0.1 min. The supernatant was separated, washed with 1.5 mL M phosphate buffer (pH 6.5), mixed with 5 mL of methy- of dichloromethane by vortexing for 30 s, and centrifuged lene chloride, and centrifuged for 2–3 min. Then 2.5 mL again. The aqueous layer was separated and assayed. Un- of the clear organic layer was separated, evaporated to der these conditions, retention times of pirenzepine and dryness at 40◦C under a stream of nitrogen, reconsti- metformin were about 4.8 and 7.1 min, respectively. tuted with 50–75 ␮L of trimethylphenylammonium hy- Calibration curves for metformin were construced over droxide, vortexed for 5 min, and assayed. Under these the range from 20 to 4000 mg/L. Correlation coefficients conditions, retention times for ethosuximide, mesuximide, were greater than 0.9998. Recovery of the drug from phenobarbital, carbamazepine, primidone, and pheny- plasma was higher than 95%. Intraday and interday co- toin were 2.48, 9.93, 11.91, 12.43, 17.56, and 19.8 min, efficients of variation were less than 5.3% and 6.4%, re- respectively. spectively. Limits of detection and quantification were A calibration curve for mesuximide was constructed 4 and 10 mg/L, respectively. over the range from 10 to 80 ␮g/mL. The average recovery of the drug from plasma was 99%. The limit of quantifi- Assay 2 AbuRuz et al. [2] described an HPLC method cation was 0.5 ␮g/mL. There was no interference with the for therapeutic drug monitoring of metformin using dried assay from kemadrin, mephenytoin, haldol, or prolixin. blood spots. A Shimadzu liquid chromatograph consisting of a model LC10ADVP pump, model SIL10ADVP autosampler, model DGE14A degasser, model SCL10ADVP controller, and model SDPM10ADVP photodiode-array de- REFERENCE tector was used. The stationary phase was a Waters Sym- metry C18 column (150 × 4.6 mm, 5 ␮m particle size) pro- 1. Varma R, Therapeutic monitoring of anticonvulsant tected by a Waters Symmetry C18 guard column. The mo- drugs in psychiatric patients: Rapid, simultaneous gas- bile phase consisted of acetonitrile and 12.5 mM monobasic chromatographic determination of six commonly used anticon- potassium phosphate buffer (37.5 : 62.5) containing 2 mM vulsants without interference from other drugs, Biochem Exp Biol 14: 311–318 (1978). sodium dodecylsulfate, adjusted to pH 7.3 with sodium hydroxide. The flow rate was 0.5 mL/min. UV detection was performed at 236 nm. A stock solution of salbutamol at 10 ␮g/mL was prepared in water. A working solution of salbutamol at 200 METFORMIN HYDROCHORIDE ng/mL was prepared by diluting the stock solution with methanol and was used as an internal standard. A stock CHEMISTRY solution of metformin at 50 ␮g/mL was prepared. Its working solutions were prepared by diluting this stock solution Metformin hydrochloride is a biguanide antidiabetic. Its with water. Standards were prepared by spiking drug-free chemical name is 1,1-dimethylbiguanide hydrochloride. human whole blood with working solutions. An aliquot of Its molecular formula is C4H11N5·HCl, with a molecular 30 ␮L of a standard was spotted directly onto a What- weight of 165.6 and a CAS number of 1115-70-4. Met- man Guthrie type card and dried overnight. A dried blood formin hydrochloride occurs as a white crystalline powder. spot of a 6-mm disk on the card was punched, mixed with Metformin hydrochloride is freely soluble in water, slightly 1 mL of 60% methanol and 20 ␮L of the internal stan- soluble in ethanol, and practically insoluble in acetone and dard solution, and vortexed for 90 s. The extract was sepa- dichloromethane. rated, evaporated to dryness at 38◦C under a stream of air, P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

METHADONE HYDROCHLORIDE 309

reconstituted in 0.2 mL of mobile phase, and assayed. Un- METHADONE HYDROCHLORIDE der these conditions, retention times of metformin and salbutamol were 5.4 and 6.7 min, respectively. CHEMISTRY Calibration curves for metformin were constructed over the range from 300 to 5000 ng/mL. Correlation coefficients Methadone is an opioid analgesic. Its chemical name were greater than 0.99. The mean accuracy expressed as is (±)-6-dimethylamino-4,4-diphenylheptan-3-one hydro- the relative percentage error was 10.7%. The mean recov- chloride. Its molecular formula is C21H27NO·HCl, with a ery of metformin from whole blood was 84.0%. Intraday molecular weight of 345.9 and a CAS number of 125-56-4. and interday coefficients of variation were less than 5.4% Methadone hydrochloride occurs as odorless colorless crys- and 8.7%, respectively. Limits of detection and quantifica- tals or white crystalline powder. Methadone hydrochloride tion were 90 and 150 ng/mL, respectively. is soluble in water and freely soluble in alcohol and chloroform. It is practically insoluble in ether and glycerol. Assay 3 Porta et al. [3] reported the determination of met- Methadone hydrochloride should be stored in airtight con- formin in human plasm by HPLC-UV. A Merck–Hitachi tainers at 25◦C and protected from light. series L7000 liquid chromatograph consisting of a model L7110 pump, model L7612 vacuum degasser, model L7200 autoinjector with variable injection valve, model L7300 column oven, and model L7400 UV–visible detector was used. METHODS The stationary phase was a MetaChem MetaSil phenyl column (150 × 4.6 mm, 4 ␮m particle size). The column Assay 1 Kintz et al. [1] reported a capillary GC method temperature was maintained at 40◦C. The mobile phase for the simultaneous screening and quantification of alfen- consisted of 0.02 M phosphate buffer (pH 7.0) and acetoni- tanil, dextromoramide bitartrate, fentanyl, methadone trile (50 : 50, vol/vol) and was isocratically delivered at hydrochloride, pentazocine hydrochloride, pethidine hy- 1.0 mL/min. UV detection was performed at 236 nm. The drochloride, phenoperidine hydrochloride, and phency- injection volume was 25 ␮L. clidine hydrochloride in human plasma. A Perkin- Working solutions of metformin were prepared in Elmer 8500 gas chromatograph was equipped with a methanol. Standards were prepared by spiking blank hu- nitrogen–phosphorous detector and a Perkin-Elmer Sigma man plasma with working solutions. A stock solution of 15 data collector. The separation of drugs was performed propranolol at 10 ␮g/mL was prepared in methanol and on a vitreous silica capillary, bonded phase BP 10 (SGE) was used as an internal standard. An aliquot of 50 ␮L column (25 m × 0.22 mm). The flow rate of carrier gas (ni- of internal standard solution was evaporated to dryness trogen) was 6.2 mL/min. The head pressure on the column at 40◦Cunderastreamofnitrogenina10-mLglass was maintained at 18 psi. The temperatures for injector tube, mixed with 500 ␮L of plasma sample or standard and detector were set at 270◦C and 280◦C, respectively. The and 50 ␮L of 0.05 M hydrochloric acid, vortexed for 30 s, column oven temperature was programmed from 200◦Cto mixed with 2 mL of acetonitrile, centrifuged at 1900 g for 270◦Cat10◦C/min and held at 270◦C for 23 min. The total 10 min, filtered through a 0.45-␮m Durapore membrane runtime of an injection was 30 min. filter, evaporated to dryness at 40◦C under a stream of Stock solutions of drugs (10 mg/L each) were prepared nitrogen, reconstituted with 300 ␮L of mobile phase, and in methanol and stored at 4◦C. SKF-525A from Smith, assayed. Under these conditions, retention times of met- Kline and French (UK) was used as an internal stan- formin and propranolol were 7.5 and 9.5 min, respectively. dard. A plasma sample (1 mL) was mixed with 20 ␮Lof4 Calibration curves for metformin were constructed over N sodium hydroxide solution, 1 mL of dibasic potassium the range from 30 to 4000 ng/mL. Correlation coeffi- phosphate buffer (40%, pH 9.2), 20 ␮L of the internal stan- cients were 0.9996. The accuracy ranged from 87.0% to dard (10 mg/L), and 4.5 mL of chloroform/isopropranol/n- 98.3%. The average recovery of metformin from plasma heptane (50 : 17 : 33, vol/vol/vol), and centrifuged. The was 93.7%. Limit of quantification was 30 ng/mL. Intra- organic phase was collected, evaporated to dryness at day and interday coefficients of variation were less than 45◦C, reconstituted in 20 ␮L of methanol, and assayed. 8.4% and 11.3%, respectively. The injection volume was 1 ␮L. Under these conditions, retention times of pethidine, norpethidine, phencyclidine, methadone metabolite, methadone, pentazocine, fentanyl, dextromoramide, and alfentanil were 5.18, 6.06, REFERENCES 6.42, 6.89, 7.59, 8.88, 19.73, 25.47, and 29.57 min, respectively. 1. Jiang Y, Mao D, Huang R, et al., An improved HPLC method for determination of metformin hydrochloride in human plasma, A standard curve for methadone was constructed by Chinese J Clin Pharm 13: 338–341 (2004). plotting peak area ratios of the drug to the internal stan- 2. AbuRuz S, Millership J, McElnay J, Dried blood spot liquid dard against the concentrations. The correlation coeffi- chromatography assay for therapeutic drug monitoring of met- cient was greater than 0.99. The extraction recovery of formin, J Chromatogr B 832: 202–207 (2006). methadone in plasma was 82.7%. Within-day and between- 3. Porta V, Schramm SG, Kano EK, et al., HPLC-UV determina- day coefficients of variation were less than 5.7% and 6.0%, tion of metformin in human plasma for application in pharma- respectively. The limit of detection was 1.3 ␮g/L. cokinetics and bioequivalence studies, J Pharm Biomed Anal This method was free of interference from endogenous 46: 143–147 (2008). plasma materials. P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

310 METHADONE HYDROCHLORIDE

Assay 2 Mercolini et al. [2] described the simultane- water, triethylamine, and glacial acetic acid (10 : 90 : ous determination of methadone, buprenorphine, and nor- 0.5 : 0.7, vol/vol/vol/vol) and was isocratically delivered at buprenorphine in biological fluids for therapeutic drug 0.175 mL/min. The injection volume was 40 ␮L. monitoring purposes. An Agilent 1100 Series liquid chro- A Shimadzu LCMS-2010A mass spectrometer equipped matograph equipped with a photodiode-array detector was with an atmospheric-pressure chemical ionization (APCI) used. The stationary phase was a Varian Microsorb-MV probe was operated in the positive-ion mode. The optimized C8 column (250 × 4.6 mm, 5 ␮m particle size). The mo- parameters were set as follows: heating block temperature bile phase was composed of methanol, acetonitrile, and 200◦C, CDL temperature 250◦C, CDL voltage 40 V, APCI 60 mM monobasic potassium phosphate buffer containing probe temperature 400◦C, and corona needle voltage 4.5 0.5% triethylamine (50 : 10 : 40, vol/vol/vol) adjusted to kV. Quantification was performed in the selected-ion mon- pH 6.0 with phosphoric acid and was isocratically deliv- itoring (SIM) mode using target ions m/z 310.15, 313.15, ered at 1.0 mL/min. UV detection was performed at 214 and 316.15 for methadone, methadone-d3, and methadone- nm. The injection volume was 50 ␮L. d6, respectively. Stock solutions of methadone at 1 mg/mL, buprenor- Methadone-d3 at 200 ng/mL was used as an internal phine at 8 mg/mL, and norbuprenorphine at 3 mg/mL were standard. Standards were prepared by spiking blank hu- separately prepared in methanol. A stock solution of loxap- man plasma with working solutions containing methadone ine at 1.0 mg/mL was prepared in methanol and was used and methadone-d6. An aliquot of 1 mL of plasma sam- as an internal standard. Working solutions were prepared ple or standard was mixed with 100 ␮Loftheinternal by diluting stock solutions with mobile phase. Standards standard solution, alkalinized by 0.4 mL of 0.1 M sodium in plasma or urine were prepared by spiking blank hu- carbonate (pH 10), extracted with 5 mL of diethyl ether man plasma or urine with working solutions. An aliquot and hexane (30 : 70, vol/vol) for 20 min on a rotary mixer, of 300 ␮L of urine was mixed with 1.5 mL of 37% (wt/wt) and centrifuged at 2000 g for 10 min. The upper organic hydrochloric acid, vortexed for 10 min, heated at 120◦Cfor phase was separated, mixed with 200 ␮L of 5 mM hy- 20 min, cooled to room temperature, adjusted to pH 7.0 drochloric acid, vortexed for 1 min, and centrifuged again. with 2 N sodium hydroxide solution, and filtered through The organic phase was discarded, and the aqueous phase a 20-␮m nylon filter. An aliquot of 300 ␮Lofplasmaorhy- was assayed. Under these conditions, retention times of drolyzed urine was mixed with 600 ␮L of water and 50 ␮L (R)-methadone and (S)-methadone were 7.1 and 8.1 min, of the internal standard solution, loaded onto an IST Iso- respectively. lute C8 solid-phase extraction cartridge (100 mg/1 mL) that Calibration curves for (R)-methadone were constructed was preconditioned with 1 mL of methanol 5 times followed over the range from 0.5 to 75 ng/mL. Correlation coeffi- by 1 mL of water 5 times, washed twice with 1 mL of wa- cients were greater than 0.99. The extraction efficiencies ter and twice with 1 mL of 20% methanol in water, dried were higher than 80%. Intraassay and interassay coeffi- under vacuum for 1 min, eluted with 1 mL of methanol, cients of variation were less than 2.6% and 3.8%, respec- again dried under vacuum, reconstituted with 150 ␮Lof tively. mobile phase, and assayed. Under these conditions, re- Calibration curves for (S)-methadone were constructed tention times of norbuprenorphine, methadone, buprenor- over the range from 0.5 to 75 ng/mL. Correlation coeffi- phine, and loxapine were 4.0, 5.1, 6.0, and 7.0 min, cients were greater than 0.99. The extraction efficiencies respectively. was higher than 78%. Intraassay and interassay coeffi- Calibration curves for methadone were constructed over cients of variation were less than 2.2% and 3.7%, respec- the range from 10.0 to 1500.0 ng/mL. Correlation coeffi- tively. cients were greater than 0.9998. The mean extraction yield Calibration curves for (R)-methadone-d6 were con- of methadone was higher than 96%. Intraday and inter- structed over the range from 0.5 to 75 ng/mL. Correla- day coefficients of variation were less than 5.5% and 6.1%, tion coefficients were greater than 0.99. The extraction respectively. Limits of detection and quantification were efficiencies was higher than 83%. Intraassay and interas- 0.4 and 1.0 ng/mL, respectively. There was no interference say coefficients of variation were less than 6.3% and 4.6%, from endogenous materials and from the following drugs: respectively. amitriptyline, citalopram, fluoxetine, imipramine, sertra- Calibration curves for (S)-methadone-d6 were con- line, venlafaxine, bromazepam, brotizolam, clonazepam, structed over the range from 0.5 to 75 ng/mL. Correlation diazepam, flurazepam, lorazepam, codeine, morphine, coefficients were greater than 0.99. The extraction effi- amphetamine, methamphetamine, MDMA (Ecstasy), 9- ciencies was higher than 81%. Intraassay and interassay THC (tetrahydrocannabinol), and 11-nor-9-carboxy-9- coefficients of variation were less than 8.1% and 4.5%, re- THC. spectively. The limit of quantification for all analytes was 0.5 ng/mL. Assay 3 Foster et al. [3] developed an LC-MS/MS method for quantification of the individual enantiomers of methadone in human plasma. A Shimadzu liquid chro- REFERENCES matograph consisting of a model LC10AD pump, model SIL10AD autoinjector, model SPD10A UV–visible detec- 1. Kintz P, Tracqui A, Lugnier AJ, et al., Simultaneous screening tor, and model DGU12A degasser was used. The stationary and quantification of several nonopiate narcotic analgesics and phase was an Astec Cyclobond I 2000 RSP column (150 × phencyclidine in human plasma using capillary gas chromatog- 2.1 mm). The mobile phase was composed of acetonitrile, raphy, Meth Find Exp Clin Pharmacol 12: 193–196 (1990). P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

METHAMPHETAMINE HYDROCHLORIDE 311

2. Mercolini L, Mandrioli R, Conti M, et al., Simultaneous deter- with water, and microextracted. For hair, 10-mg samples mination of methadone, buprenorphine and norbuprenorphine were cleaned, dried, cut into small pieces (0.5 cm), sub- in biological fluids for therapeutic drug monitoring purposes, merged in 5 mL of methanol and 5 M hydrochloric acid J Chromatogr B 847: 95–102 (2007). (20 : 1, vol/vol), sonicated for 1 h, allowed to stand at 3. Foster, DJR, Morton EB, Biotech B, et al., Stereoselective room temperature overnight, and filtered. The filtrate was quantification of methadone and a d6-labeled isotopomer using evaporated to dryness under a stream of nitrogen, redis- high performance liquid chromatography–atomospheric pres- solved in 1 mL of 100 mM carbonate buffer (pH 10.0), sure chemical ionization mass-spectrometry: Application to a and microextracted. A laboratory-made polypyrrole (PPY)- pharmacokinetic study in a methadone maintained subject, Ther Drug Monit 28: 559–567 (2006). coated capillary (60 cm long) was used as the in-tube SPME device, which was conditioned with 2 × 40 ␮Lof methanol followed by 2 × 40 ␮L of water and placed between the injection needle and the loop of the autosam- METHAMPHETAMINE HYDROCHLORIDE pler. An aliquot of 40 ␮L of the diluted urine sample or a hair filtrate was drawn from a sample vial into the cap- CHEMISTRY illary at 100 ␮L/min and then ejected back to the sample vial. This draw/eject process was repeated 10 times. Af- Methamphetamine hydrochloride is a central stimulant. ter the tip of the injection needle was washed with 2 ␮L Its chemical name is (+)-N,␣-dimethylphenethylamine hy- of methanol and the valve was switched to the analytical drochloride. Another name is metamfetamine hydrochlo- column from the load postion, extracted drugs were des- ride. Its molecular formula is C10H15N·HCl, with a molec- orbed from the capillary coating with mobile phase and ular weight of 185.7 and a CAS number of 51-57-0. assayed. Under these conditions, retention times of am- Methamphetamine hydrochloride occurs as white, odor- phetamine, 3,4-methylenedioxyamphetamine, metham- less or practically odorless crystals or crystalline powder. phetamine, 3,4-methylenedioxymethamphetamine, and Methamphetamine hydrochloride is freely soluble in wa- 3,4-methylenedioxyethylamphetamine were about 3.4, 3.7, ter, ethanol, and chloroform. It should be stored in airtight 4.1, 4.6, and 5.0 min, respectively (estimated from the pub- containers and protected from light. lished chromatogram). Calibration curves for methamphetamine were constructed over the range from 0.1 to 100 ng/mL in water. METHODS Correlation coefficients were greater than 0.9998. Within- day and between-day coefficients of variation were 1.2% Assay 1 Wu et al. [1] described the determina- and 3.2%, respectively. The limit of detection was 8 ng/L. tion of the stimulants amphetamine, metham- Calibration curves for methamphetamine were also con- phetamine, 3,4-methylenedioxyamphetamine, 3,4-methyl- structed over the range from 0.5 to 100 ng/mL in urine. enedioxymethamphetamine, and 3,4-methylenedioxy- Correlation coefficients were greater than 0.9996. The re- ethylamphetamine in human urine and hair samples covery was greater than 94.7%. Within-day and between- by polypyrrole-coated capillary in-tube solid-phase mi- day coefficients of variation were 2.2% and 3.0%, respec- croextraction (SPME) coupled with liquid chromat- tively. The limit of quantitation was 4 ng/mL. Calibra- ography–electrospray mass spectrometry. An Agilent tion curves for methamphetamine were constructed over 1100 Series liquid chromatograph was used. The sta- the range from 1 to 100 ng/mL in hair samples. Correla- tionary phase was a Supelcosil LCCN column (330 × tion coefficients were greater than 0.9997. Within-day and 4.6 mm, 3 ␮m particle size). The mobile phase consisted between-day coefficients of variation were 0.8% and 2.5%, of acetonitrile and 50 mM ammonium acetate buffer respectively. The limit of quantitation was 0.25 ng/10 mg (15 : 85) and was isocractically delivered at 0.4 mL/min. hair. An Agilent mass spectrometer equipped with an atmospheric-pressure–electrospray ionization interface Assay 2 Stanaszek and Piekoszewski [2] reported the was operated in the positive ionization mode. The op- simultaneous determination of amphetamine (AMP), timal conditions were as follows: nebulizer gas (ni- ephedrine (EP), methcathinone (MTC), p-methoxy- trogen) 40 psi, drying gas (nitrogen) 12 L/min and amphetamine (PMA), methylenedioxyamphetamine 350◦C, capillary voltage 1 kV, dwell time 78 ms, and (MDA), methylenedioxymethamphetamine (MDMA), fragmenter voltages 30 V for amphetamine and 3,4- methylenedioxyethylamphetmaine (MDEA), and metham- methylenedioxyamphetamine and 50 V for methamphetamine (MA) in human hair by high-performance phetamine, 3,4-methylenedioxymethamphetamine, and liquid chromatography–atmospheric-pressure chemi- 3,4-methylenedioxyethylamphetamine. Drugs were quan- cal ionization mass spectrometry (HPLC-APCI-MS). tified in selected-ion monitoring (SIM) mode at m/z 136 A Hewlett-Packard 1100 Series liquid chromatograph for amphetamine, m/z 150 for methamphetamine, m/z coupled to a mass spectrometer equipped with APCI 180 for 3,4-methylenedioxyamphetamine, m/z 194 for 3,4- interface was used. The stationary phase was a Merck methylenedioxymethamphetamine, and m/z 208 for 3,4- LiChroCART Purospher 60 RP18e column (125 × 4.0 mm, methylenedioxyethylamphetamine. 5 ␮m particle size) protected by a RP18e guard column. Urine samples were diluted 10 times with water. An The column temperature was maintained at 35◦C. The aliquot of the diluted urine sample was mixed with 0.2 mL mobile phase consisted of 0.1% formic acid in acetonitrile of 0.5 M sodium carbonate buffer (pH 10.0), filled to 1 mL (A) and 0.1% formic acid in water (B) and was delivered P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

312 METHCATHINONE

at 1 mL/min in a gradient mode as follows: 100% A at 0 METHCATHINONE min, 60% A at 15 min, 100% A at 15.2 min, and 100% A at 20 min. The total runtime was 20 min. The mass CHEMISTRY spectrometer was operated in the positive-ion mode under the following optimal operating parameters: fragmenter Methcathinone is a stimulant. Its chemical name is 2- voltage 50 V, capillary voltage 3.3 kV, corona current 4 (methylamino)-1-phenyl-1-propan-1-one. Other name in- ␮ ◦ A, drying gas temperature 280 C, vaporizer temperature cludes ephedrone. Its molecular formula is C10H13NO, with 320◦C, drying gas flow 3 L/min, and nebulizer (nitrogen) a molecular weight of 163.2 and a CAS number of 5650- pressure 30 psi. Analytes were detected in the selected-ion 44-2. monitoring (SIM) mode at following ions: m/z 166.2 for EP, 164.2 for MTC, 166.2 for PMA,136.2 for AMP, 150.2 for MA, 180.2 for MDA, 194.2 for MDMA, 208.3 for MDEA, METHOD 169.2 for EP-d3, 141.2 for AMP-d5, 155.2 for MA-d5, 185.2 for MDA-d5, 199.2 for MDMA-d5, and 213.3 for MDEA-d5. Assay 1 Stanaszek and Piekoszewski [1] reported the Stock solutions of AMP, EP, MTC, PMA, MDA, MDMA, simultaneous determination of amphetamine (AMP), andMDEAat1mg/mLandMA,AMP-d5,EP-d3,MA-d5, ephedrine (EP), methcathinone (MTC), paramethoxyam- MDA-d5,MDMA-d5, and MDEA-d5 at 0.1 mg/mL were pre- phetamine (PMA), methylenedioxyamphetamine (MDA), pared in methanol. Deuterated amphetamines were used methylenedioxymethamphetamine (MDMA), methylene- as internal standards. Working solutions were prepared dioxyethylamphetmaine (MDEA), and methamphetamine by diluting stock solutions with water. Stock and working (MA) in human hair by high-performance liquid solutions were stored at −20◦C. Standards were prepared chromatography–atmospheric-pressure chemical ioniza- by spiking drug-free samples with working solutions. tion mass spectrometry (HPLC-APCI-MS). A Hewlett- In this assay, 50 mg of hair sample was washed succes- Packard 1100 series liquid chromatograph coupled to a sively by sonication in 15 mL of dichloromethane for 5 min, mass spectrometer equipped with an APCI interface was 15 mL of water for 5 min, and 15 mL of methanol for 5 used. The stationary phase was a Merck LiChroCART min, dried at room temperature; cut into 2-cm segments, Purospher 60 RP18e column (125 × 4.0 mm, 5 ␮m par- then cut into 1-mm pieces; pulverized; spiked with 25 ␮L ticle size) protected by a RP18e guard column. The column of internal standard; hydrolyzed in 1 mL of 1 M sodium temperature was maintained at 35◦C. The mobile phase hydroxide; incubated at 70◦C for 20 min; cooled down to consisted of 0.1% formic acid in acetonitrile (A) and 0.1% room temperature; extracted with 2 mL of 1-chlorobutane formic acid in water (B) and was delivered at 1 mL/min for 10 min; and centrifuged at 4000 rpm for 5 min. The or- in a gradient mode as follows: 100% A at 0 min, 60% A at ganic layer was collected and acidified with 100 ␮L of 0.025 15 min, 100% A at 15.2 min, and 100% A at 20 min. The to- M hydrochloric acid. The organic solvent was evaporated tal runtime was 20 min. The mass spectrometer was oper- at 40◦C under a stream of nitrogen and assayed. Under ated in the positive-ion mode under the following optimal these conditions, retention times in minutes were 5.79 for operating parametes: fragmenter voltage 50 V, capillary EP, 7.10 for MTC, 7.35 for PMA, 6.53 for AMP, 7.10 for MA, voltage 3.3 kV, corona current 4 ␮A, drying gas tempera- 7.02 for MDA, 7.38 for MDMA, 8.18 for MDEA, 5.77 for EP- ture 280◦C, vaporizer temperature 320◦C, drying gas flow d3,6.48forAMP-d5, 7.08 for MA-d5,6.98forMDA-d5, 7.42 3 L/min, and nebulizer (nitrogen) pressure 30 psi. Ana- for MDMA-d5, and 8.16 for MDEA-d5. lytes were detected in the selected-ion monitoring (SIM) Calibration curves for methamphetamine were con- mode at the following ions: m/z 166.2 for EP, 164.2 for structed in the range from 0.2 to 20.0 ng/mg. Correlation MTC, 166.2 for PMA,136.2 for AMP, 150.2 for MA, 180.2 coefficients were 0.999. The recovery of methamphetamine for MDA, 194.2 for MDMA, 208.3 for MDEA, 169.2 for EP- from hair ranged from 58.4% to 85.5%. The accuracy d3, 141.2 for AMP-d5, 155.2 for MA-d5, 185.2 for MDA-d5, ranged from 97.0% to 100.5%. Intraday and interday co- 199.2 for MDMA-d5, and 213.3 for MDEA-d5. efficients of variation were 11.4% and 13.7%, respectively. Stock solutions of AMP, EP, MTC, PMA, MDA, MDMA, Limits of detection and limit of quantification were 0.05 and MDEA at 1 mg/mL and MA, AMP-d5,EP-d3,MA-d5, and 0.10 ng/mg, respectively. MDA-d5,MDMA-d5, and MDEA-d5 at 0.1 mg/mL were prepared in methanol. Deuterated amphetamines were used as internal standards. Working solutions were prepared REFERENCE by diluting stock solutions with water. Stock and working solutions were stored at −20◦C. Standards were prepared 1. Wu J, Lord H, Pawliszyn J, Determination of stimulants in by spiking drug-free samples with working solutions. human urine and hair samples by polypyrrole coated capil- In this assay 50 mg of hair sample was washed succes- lary in-tube solid phase microextraction coupled with liquid sively by sonication in 15 mL of dichloromethane for 5 min, chromatography–electrospray mass spectrometry, Talanta 54: 655–672 (2001). 15 mL of water for 5 min, and 15 mL of methanol for 5 min; dried at room temperature; cut into 2-cm segments, then 2. Stanaszek R, Piekoszewski W, Simultaneous determina- ␮ tion of eight underivatized amphetamines in hair by high- cut into 1-mm pieces; pulverized; spiked with 25 Lof performance liquid chromatography–atmospheric pressure internal standard; hydrolyzed in 1 mL of 1 M sodium hy- ◦ chemical ionization mass spectrometry (HPLC-APCI-MS), droxide; incubated at 70 C for 20 min, cooled down to room J Anal Toxicol 28: 77–85 (2004). temperature; extracted with 2 mL of 1-chlorobutane for 10 min; and centrifuged at 4000 rpm for 5 min. The organic P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

METHYCLOTHIAZIDE 313

layer was collected and acidified with 100 ␮L of 0.025 M using linear regression analysis and Bland–Altaman plots hydrochloric acid. The organic solvent was evaporated at to assess bias. A relationship between these two meth- 40◦C under a stream of nitrogen and assayed. Under these ods was obtained: [TDx FLx FPIA] = 0.941 ∗ [Dimension conditions, retention times in minutes were 5.79 for EP, RxL Max]–0.039 (Syx = 0.297, r = 0.992, n = 71). Po- 7.10 for MTC, 7.35 for PMA, 6.53 for AMP, 7.10 for MA, tential crossreactants such as dihydrofolic acid, folic acid, 7.02 for MDA, 7.38 for MDMA, 8.18 for MDEA, 5.77 for methopterin, and trimethoprim, did not cause a difference EP-d3,6.48forAMP-d5,7.08forMA-d5,6.98forMDA-d5, greater than 10% of the blank at the methotrexate concen- 7.42 for MDMA-d5, and 8.16 for MDEA-d5, respectively. tration level of 1.0 ␮M. Aminopterin interfered with this Calibration curves for methcathinone were constructed assay. in the range from 0.2 to 20.0 ng/mg. Correlation coefficients were 0.999. The recovery of methcathinone from hair ranged from 69.8% to 94.4%. The accuracy ranged REFERENCE from 86.0% to 111.6%. Intraday and interday coefficients of variation were 7.6% and 19.0%. Limits of detection and 1. Mendu DR, Chou PP, Soldin SJ, An improved application for quantification were 0.20 and 0.30 ng/mg, respectively. the enzyme multipled immunoassay technique for caffeine, amikacin, and methotrexate assays on the Dade-Behring Di- mension RxL Max clinical chemistry system, Ther Drug Monit REFERENCE 29: 632–637 (2007).

1. Stanaszek R, Piekoszewski W, Simultaneous determination of eight underivatized amphetamines in hair by high- performance liquid chromatography–atmospheric pressure METHYCLOTHIAZIDE chemical ionization mass spectrometry (HPLC-APCI-MS), J Anal Toxicol 28: 77–85 (2004). CHEMISTRY

Methyclothiazide is a thiazide diuretic. Its chemical name is 6-chloro-3-chloromethyl-3,4-dihydro-2-methyl-2H-1,2,4- METHOTREXATE benzothiadiazine-7-sulfonamide 1,1-dioxide. Its molecular formula is C9H11Cl2N3O4S2, with a molecular weight of CHEMISTRY 360.2 and a CAS number of 135-07-9. Methyclothiazide occurs as a white or near-white, odorless or slightly odorous, Methotrexate is an antineoplastic. Its chemical name white crystalline powder. Methyclothiazide is very slightly is N-{4-[(2,4-diamino-6-pteridinylmethyl) methylamino] soluble to practically insoluble in water and chloroform benzoyl}-L-glutamic acid. Its molecular formula is and sparingly soluble in methanol. It is soluble 1 in 92.5 of C H N O , with a molecular weight of 454.4 and a CAS 20 22 8 5 alcohol and 1 in 2700 of ether and freely soluble in acetone number of 59-05-2. Methotrexate occurs as a yellow or and pyridine. The drug has a pK of 9.4. orange-brown crystalline powder. Methotrexate is practi- a cally insoluble in water, ethanol, chloroform, and ether. It is freely soluble in dilute solutions of alkali hydroxides and carbonates. Methotrexate should be stored in airtight METHOD containers and protected from light. Assay 1 Fullinfaw et al. [1] described an HPLC method for screening chlorothiazide, hydrochloroth- METHOD iazide, quinethazone, chlorthalidone, methyclothiazide, clopamide, frusemide, metolazone, mefruside, bendroflu- Assay 1 Mendu et al. [1] reported an improved appli- azide, cyclopenthiazide, and bumetanide in human urine. cation of the enzyme multiple-immunoassay technique A Hewlett-Packard 1090A liquid chromatograph equipped (EMIT) for methotrexate. A Dade–Behring Dimension RxL with a photodiode array detector was used. The stationary Max automated clinical analyzer was used. Calibrators for phase was a Merck LiChrosorb RP18 column (125 × 4 mm, methotrexate were reconstituted according to the manu- 5 ␮m particle size). The column temperature was main- facturer’s instuctions. Reagent 1 (R1) and reagent 2 (R2) tained at 50◦C. The mobile phase consisted of acetonitrile were separately dissolved with 3 mL of distilled water, and and 0.01 M phosphate buffer (pH 3.0) and was delivered at EMIT drug assay buffer concentrate was diluted with dis- 1.5 mL/min in a gradient mode. The acetonitrile content of tilled water (1 : 14, vol/vol). One part of R1 and R2 was mobile phase was increased from 10% at 1.5 min to 35% at then separately mixed with 7 parts of EMIT drug assay 3.5 min. The total runtime was 10 min. UV detection was buffer. The optical density changes were monitored for a performed at 271 nm. The injection volume was 5 ␮L. longer period of time than recommended by the manufac- Stock solutions of chlorothiazide, hydrochloroth- turer. iazide, quinethazone, chlorthalidone, methyclothiazide, A linear calibration curve was constructed over the clopamide, frusemide, metolazone, mefruside, bendroflu- range from 0 to 1 ␮M. Within-day and between-day coeffi- azide, cyclopenthiazide, and bumetanide at 1 mg/mL were cients of variation were less than 11.4% and 12.3%, respec- separately prepared in ethanol and stored at 4◦C. Working tively.Results obtained by Dimension RxL Max EMIT were solutions of these drugs were prepared by diluting stock compared with those obtained by Abbott TDx FLx FPIA solutions with ethanol. Standards were prepared by P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

314 3,4-METHYLENEDIOXYAMPHETAMINE

spiking blank human urine with working solutions. An The mobile phase consisted of acetonitrile and 50 mM am- aliquot of 2 mL of urine samples was acidified with 2 monium acetate buffer (15 : 85) and was isocractically de- mL of 1 M monobasic sodium phosphate buffer (pH 4.1), livered at 0.4 mL/min. mixed with 4 mL of ethyl acetate, vortexed for 2 min, and An Agilent mass spectrometer equipped with an centrifuged at 1500 g for 5 min. The organic layer was atmospheric-pressure–electrospray ionization interface separated, mixed with 5 mL of 0.1 M dibasic sodium phos- was operated in the positive ionization mode. The op- phate buffer (pH 7.5), vortexed for 2 min, and centrifuged. timal conditions were as follows: nebulizer gas (ni- The organic phase was collected, evaporated to dryness at trogen) 40 psi, drying gas (nitrogen) 12 L/min and 60◦C under nitrogen, reconstituted with 100 ␮L of 35% ace- 350◦C, capillary voltage 1 kV, dwell time 78 ms, and tonitrile in 0.01 M phosphate buffer (pH 3.0), and assayed. fragmenter voltages 30 V for amphetamine and 3,4- Under these conditions, retention times for chlorothiazide, methylenedioxyamphetamine and 50 V for metham- hydrochlorothiazide, quinethazone, chlorthalidone, phetamine, 3,4-methylenedioxymethamphetamine, and methyclothiazide, frusemide, metolazone, mefruside, 3,4-methylenedioxyethylamphetamine. Drugs were quan- bendrofluazide, cyclopenthiazide, and bumetanide were tified in selected-ion monitoring (SIM) mode at m/z 136 about 1.9, 2.2, 3.4, 4.2, 4.7, 5.1, 5.3, 6.2, 6.8, 7.2, and 9.3 for amphetamine, m/z 150 for methamphetamine, m/z min, respectively (estimated from the published chro- 180 for 3,4-methylenedioxyamphetamine, m/z 194 for 3,4- matogram). Clopamide had the same retention time as methylenedioxymethamphetamine, and m/z 208 for 3,4- did methyclothiazide. These two drugs were identified by methylenedioxyethylamphetamine. comparing the peak UV spectra between 220 and 400 nm. Urine samples were diluted 10 times with water. An The mean recovery of methylclothiazide from urine aliquot of the diluted urine sample was mixed with 0.2 was 87%. There was no interference with this screen- mL of 0.5 M sodium carbonate buffer (pH 10.0), filled to ing procedure from the following drugs: acetylsalicylic 1 mL with water, and microextracted. For hair samples, acid, allopurinol, alprenolol, atenolol, captopril, carbima- 10 mg of hair were cleaned, dried, cut into small pieces zole, clonidine, coloxyl, danthron, diazepam, digoxin, dox- (0.5 cm), submerged in 5 mL of methanol and 5 M hy- epin, glibenclamide, hydralazine, indomethacin, labetalol, drochloric acid (20 : 1, vol/vol), sonicated for 1 h, allowed metformin, methyldopa, metoprolol, mianserin, minoxidil, to stand at room temperature overnight, and filtered. The nifedipine, nitrazepam, oxazepam, oxprenolol, pindolol, filtrate was evaporated to dryness under a stream of nitro- prazosin, proparanolol, salbutamol, senokot, theophylline, gen, redissolved in 1 mL of 100 mM carbonate buffer (pH and trifluoperazine. 10.0), and microextracted. A laboratory-made polypyrrole (PPY)-coated capillary (60 cm long) was used as the in- tube SPME device, which was conditioned with 2 × 40 ␮L REFERENCE of methanol followed by 2 × 40 ␮L of water and placed between the injection needle and the loop of the autosam- 1. Fullinfaw RO, Bury RW, Moulds RFW, Liquid chromato- ␮ graphic screening of diuretics in urine, J Chromatogr 415: pler. An aliquot of 40 L of the diluted urine sample or a 347–356 (1987). hair filtrate was drawn from a sample vial into the capillary at 100 ␮L/min and then ejected back to the sample vial. This draw/eject process was repeated 10 times. Af- ter the tip of the injection needle was washed with 2 ␮L 3,4-METHYLENEDIOXYAMPHETAMINE of methanol and the valve was switched to the analytical column from the load postion, extracted drugs were des- CHEMISTRY orbed from the capillary coating with mobile phase and assayed. Under these conditions, retention times of am- 3,4-Methylenedioxyamphetamine is a stimulant. phetamine, 3,4-methylenedioxyamphetamine, metham- Its chemical name is ␣-methyl-3,4-methylenedioxy- phetamine, 3,4-methylenedioxymethamphetamine, and phenethylamine. Another name is tenamfetamine. Its 3,4-methylenedioxyethylamphetamine were about 3.4, 3.7, molecular formula is C H NO , with a molecular weight 10 13 2 4.1, 4.6, and 5.0 min, respectively (estimated from the pub- of 179.2 and a CAS number of 4764-17-4. lished chromatogram). Calibration curves for 3,4-methylenedioxyampheta- METHODS mine were constructed over the range from 0.1 to 100 ng/mL in water. Correlation coefficients were greater Assay 1 Wu et al. [1] described the determination of than 0.9998. Within-day and between-day coefficients of stimulants, amphetamine, methamphetamine, 3,4-methy- variation were 2.6% and 4.5%, respectively. The limit lenedioxyamphetamine, 3,4-methylenedioxymethamphet- of detection was 56 ng/L. Calibration curves for 3,4- amine, and 3,4-methylenedioxyethylamphetamine in methylenedioxyamphetamine were also constructed over human urine and hair samples by polypyrrole-coated the range from 0.5 to 100 ng/mL in urine. Correlation capillary in-tube solid-phase microextraction (SPME) coefficients were greater than 0.9992. The recovery was coupled with liquid chromatography–electrospray mass greater than 89.5%. Within-day and between-day coeffi- spectrometry. An Agilent 1100 Series liquid chromato- cients of variation were 3.6% and 6.8%, respectively. The graph was used. The stationary phase was a Supelcosil limit of quantitation was 28 ng/mL. Calibration curves for LC–CN column (330 × 4.6 mm, 3 ␮m particle size). 3,4-methylenedioxyamphetamine were constructed over P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

3,4-METHYLENEDIOXYMETHAMPHETAMINE 315

the range from 1 to 100 ng/mL in hair samples. Correla- EP-d3,6.48forAMP-d5,7.08forMA-d5,6.98forMDA-d5, tion coefficients were greater than 0.9992. Within-day and 7.42 for MDMA-d5, and 8.16 for MDEA-d5, respectively. between-day coefficients of variation were 6.3% and 8.0%, Calibration curves for methylenedioxyamphetamine respectively. The limit of quantitation was 0.85 ng/10 mg were constructed in the range from 0.2 to 20.0 ng/mg. hair. Correlation coefficients were 0.999. The recovery of methylenedioxyamphetamine from hair ranged from Assay 2 Stanaszek and Piekoszewski [2] reported the 66.7% to 98.5%. The accuracy ranged from 94.9% to simultaneous determination of amphetamine (AMP), 101.0%. Intraday and interday coefficients of variation ephedrine (EP), methcathinone (MTC), p-methoxyamphet- were 4.4% and 10.0%, respectively. Limits of detection and amine (PMA), methylenedioxyamphetamine (MDA), quantification were 0.05 and 0.10 ng/mg, respectively. methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetmaine (MDEA), and methamphetamine (MA) in human hair by high-performance liquid REFERENCE chromatography–atmospheric-pressure chemical ionization mass spectrometry (HPLC-APCI-MS). A Hewlett- 1. Wu J, Lord H, Pawliszyn J, Determination of stimulants in Packard 1100 Series liquid chromatograph coupled to a human urine and hair samples by polypyrrole coated capil- mass spectrometer equipped with an APCI interface was lary in-tube solid phase microextraction coupled with liquid used. The stationary phase was a Merck LiChroCART chromatography–electrospray mass spectrometry, Talanta 54: Purospher 60 RP18e column (125 × 4.0 mm, 5 ␮m par- 655–672 (2001). ticle size) protected by a RP18e guard column. The column 2. Stanaszek R, Piekoszewski W, Simultaneous determina- temperature was maintained at 35◦C. The mobile phase tion of eight underivatized amphetamines in hair by high- consisted of 0.1% formic acid in acetonitrile (A) and 0.1% performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS), formic acid in water (B) and was delivered at 1 mL/min in J Anal Toxicol 28: 77–85 (2004). a gradient mode as follows: 100% A at 0 min, 60% A at 15 min, 100% A at 15.2 min, and 100% A at 20 min. The total runtime was 20 min. The mass spectrometer was operated in the positive-ion mode under the following optimal operating parametes: fragmenter voltage 50 V, capillary 3,4-METHYLENEDIOXYMETHAMPHETAMINE voltage 3.3 kV, corona current 4 ␮A, drying gas tempera- ◦ ◦ ture 280 C, vaporizer temperature 320 C, drying gas flow CHEMISTRY 3 L/min, and nebulizer (nitrogen) pressure 30 psi. Ana- lytes were detected in the selected-ion monitoring (SIM) 3,4-Methylenedioxymethamphetamine is a stimulant. mode at the following ions: m/z 166.2 for EP, 164.2 for Its chemical name is N,␣-dimethyl-1,3-benzodioxole-5- MTC, 166.2 for PMA,136.2 for AMP, 150.2 for MA, 180.2 ethanamine. Other names include Ecstasy and MDMA. Its for MDA, 194.2 for MDMA, 208.3 for MDEA, 169.2 for EP- molecular formula is C11H15NO2, with a molecular weight d3, 141.2 for AMP-d5, 155.2 for MA-d5, 185.2 for MDA-d5, of 193.2 and a CAS of 42542-10-9. 199.2 for MDMA-d5, and 213.3 for MDEA-d5. Stock solutions of AMP, EP, MTC, PMA, MDA, MDMA, and MDEA at 1 mg/mL and MA, AMP-d5,EP-d3,MA-d5, METHODS MDA-d5,MDMA-d5, and MDEA-d5 at 0.1 mg/mL were prepared in methanol. Deuterated amphetamines were used Assay 1 Wu et al. [1] described the determina- as internal standards. Working solutions were prepared tion of the stimulants amphetamine, methamphet- by diluting stock solutions with water. Stock and working amine, 3,4-methylenedioxyamphetamine, 3,4-methy- solutions were stored at −20◦C. Standards were prepared lenedioxymethamphetamine, and 3,4-methylenedioxy- by spiking drug-free samples with working solutions. ethylamphetamine in human urine and hair samples In this assay, 50 mg of hair sample was washed succes- by polypyrrole (PPY)-coated capillary in-tube solid- sively by sonication in 15 mL of dichloromethane for 5 min, phase microextraction (SPME) coupled with liquid 15 mL of water for 5 min, and 15 mL of methanol for 5 min, chromatography–electrospray mass spectrometry. An dried at room temperature, cut into 2-cm segments, then Agilent 1100 Series liquid chromatograph was used. The cut into 1-mm pieces, pulverized, spiked with 25 ␮Lof stationary phase was a Supelcosil LC CN column (330 × internal standard, hydrolyzed in 1 mL of 1 M sodium hy- 4.6 mm, 3 ␮m particle size). The mobile phase consisted droxide, incubated at 70◦C for 20 min, cooled down to room of acetonitrile and 50 mM ammonium acetate buffer temperature, extracted with 2 mL of 1-chlorobutane for 10 (15 : 85) and was isocractically delivered at 0.4 mL/min. min, and centrifuged at 4000 rpm for 5 min. The organic An Agilent mass spectrometer equipped with an layer was collected and acidified with 100 ␮L of 0.025 M atmospheric-pressure–electrospray ionization interface hydrochloric acid. The organic solvent was evaporated at was operated in the positive ionization mode. The op- 40◦C under a stream of nitrogen and assayed. Under these timal conditions were as follows: nebulizer gas (ni- conditions, retention times in minutes were 5.79 for EP, trogen) 40 psi, drying gas (nitrogen) 12 L/min and 7.10 for MTC, 7.35 for PMA, 6.53 for AMP, 7.10 for MA, 350◦C, capillary voltage 1 kV, dwell time 78 ms, 7.02 for MDA, 7.38 for MDMA, 8.18 for MDEA, 5.77 for and fragmenter voltages 30 V for amphetamine and P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

316 3,4-METHYLENEDIOXYMETHAMPHETAMINE

3,4-methylenedioxyamphetamine and 50 V for metham- methylenedioxyethylamphetmaine (MDEA), and methamphetamine, 3,4-methylenedioxymethamphetamine, and phetamine (MA) in human hair by high-performance 3,4-methylenedioxyethylamphetamine. Drugs were quan- liquid chromatography–atmospheric pressure chemi- tified in selected-ion monitoring (SIM) mode at m/z 136 cal ionization mass spectrometry (HPLC-APCI-MS). A for amphetamine, m/z 150 for methamphetamine, m/z Hewlett-Packard 1100 Series liquid chromatograph cou- 180 for 3,4-methylenedioxyamphetamine, m/z 194 for 3,4- pled to mass spectrometer equipped with APCI interface methylenedioxymethamphetamine, and m/z 208 for 3,4- was used. The stationary phase was a Merck LiChroCART methylenedioxyethylamphetamine. Purospher 60 RP18e column (125 × 4.0 mm, 5 ␮m particle Urine samples were diluted 10 times with water. An size) protected by a RP18e guard column. The column aliquot of the diluted urine sample was mixed with 0.2 mL temperature was maintained at 35◦C. The mobile phase of 0.5 M sodium carbonate buffer (pH 10.0), filled to 1 was composed of 0.1% formic acid in acetonitrile (A) mL with water, and microextracted. For hair samples, 10 and 0.1% formic acid in water (B) and was delivered at mg of hair was cleaned, dried, cut into small pieces (0.5 1 mL/min in a gradient mode as follows: 100% A at 0 cm), submerged in 5 mL of methanol and 5 M hydrochloric min, 60% A at 15 min, 100% A at 15.2 min, and 100% acid (20 : 1, vol/vol), sonicated for 1 h, allowed to stand A at 20 min. The total runtime was 20 min. The mass at room temperature overnight, and filtered. The filtrate spectrometer was operated in the positive-ion mode under was evaporated to dryness under a stream of nitrogen, re- the following optimal operating parametes: fragmenter dissolved in 1 mL of 100 mM carbonate buffer (pH 10.0), voltage 50 V, capillary voltage 3.3 kV, corona current 4 and microextracted. A laboratory-made polypyrrole (PPY)- ␮A, drying gas temperature 280◦C, vaporizer temperature coated capillary (60 cm long) was used as the in-tube 320◦C, drying gas flow 3 L/min, and nebulizer (nitrogen) SPME device, which was conditioned with 2 × 40 ␮Lof pressure 30 psi. Analytes were detected in the selected-ion methanol followed by 2 × 40 ␮L of water and placed be- monitoring (SIM) mode at the following ions: m/z 166.2 tween the injection needle and the loop of the autosam- for EP, 164.2 for MTC, 166.2 for PMA,136.2 for AMP, pler. An aliquot of 40 ␮L of the diluted urine sample or a 150.2 for MA, 180.2 for MDA, 194.2 for MDMA, 208.3 hair filtrate was drawn from a sample vial into the cap- for MDEA, 169.2 for EP-d3, 141.2 for AMP-d5, 155.2 for illary at 100 ␮L/min and then ejected back to the sample MA-d5, 185.2 for MDA-d5, 199.2 for MDMA-d5, and 213.3 vial. This draw/eject process was repeated 10 times. Af- for MDEA-d5. ter the tip of the injection needle was washed with 2 ␮L Stock solutions of AMP, EP, MTC, PMA, MDA, MDMA, of methanol and the valve was switched to the analytical and MDEA at 1 mg/mL and MA, AMP-d5,EP-d3,MA-d5, column from the load postion, extracted drugs were des- MDA-d5,MDMA-d5, and MDEA-d5 at 0.1 mg/mL were pre- orbed from the capillary coating with mobile phase and pared in methanol. Deuterated amphetamines were used assayed. Under these conditions, retention times of am- as internal standards. Working solutions were prepared phetamine, 3,4-methylenedioxyamphetamine, metham- by diluting stock solutions with water. Stock and working phetamine, 3,4-methylenedioxymethamphetamine, and solutions were stored at −20◦C. Standards were prepared 3,4-methylenedioxyethylamphetamine were about 3.4, 3.7, by spiking drug-free samples with working solutions. 4.1, 4.6, and 5.0 min, respectively (estimated from the pub- In this assay, 50 mg of hair sample was washed suc- lished chromatogram). cessively by sonication in 15 mL of dichloromethane for 5 Calibration curves for 3,4-methylenedioxymetha- min, 15 mL of water for 5 min, and 15 mL of methanol for mphetamine were constructed over the range from 0.1 to 5 min; dried at room temperature; cut into 2-cm segments; 100 ng/mL in water. Correlation coefficients were greater then cut into 1-mm pieces, pulverized; spiked with 25 ␮L than 0.9995. Within-day and between-day coefficients of internal standard; hydrolyzed in 1 mL of 1 M sodium of variation were 0.8% and 2.4%, respectively. The limit hydroxide; incubated at 70◦C for 20 min; cooled down to of detection was 34 ng/L. Calibration curves for 3,4- room temperature; extracted with 2 mL of 1-chlorobutane methylenedioxymethamphetamine were also constructed for 10 min; and centrifuged at 4000 rpm for 5 min. The over the range from 0.5 to 100 ng/mL in urine. Correlation organic layer was collected and acidified with 100 ␮Lof coefficients were greater than 0.9998. The recovery was 0.025 M hydrochloric acid. The organic solvent was evap- greater than 92.2%. Within-day and between-day coeffi- orated at 40◦C under a stream of nitrogen and assayed. cients of variation were 2.7% and 4.2%, respectively. The Under these conditions, retention times in minutes were limit of quantitation was 15 ng/mL. Calibration curves for 5.79 for EP, 7.10 for MTC, 7.35 for PMA, 6.53 for AMP, 7.10 3,4-methylenedioxymethamphetamine were constructed for MA, 7.02 for MDA, 7.38 for MDMA, 8.18 for MDEA, over the range from 1 to 100 ng/mL in hair samples. Cor- 5.77 for EP-d3,6.48forAMP-d5,7.08forMA-d5, 6.98 relation coefficients were greater than 0.9998. Within-day for MDA-d5,7.42forMDMA-d5, and 8.16 for MDEA-d5, and between-day coefficients of variation were 2.1% and respectively. 4.4%, respectively. The limit of quantitation was 0.65 Calibration curves for methylenedioxymetham- ng/10 mg hair. phetamine were constructed in the range from 0.2 to 20.0 ng/mg. Correlation coefficients were 0.999. The recovery Assay 2 Stanaszek and Piekoszewski [2] reported the of methylenedioxymethamphetamine from hair ranged simultaneous determination of amphetamine (AMP), from 67.1% to 91.5%. The accuracy ranged from 97.2% to ephedrine (EP), methcathinone (MTC), p-methoxy- 108.0%. Intraday and interday coefficients of variation amphetamine (PMA), methylenedioxyamphetamine were 1.6% and 4.2%, respectively. Limits of detection and (MDA), methylenedioxymethamphetamine (MDMA), quantification were 0.05 and 0.10 ng/mg, respectively. P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

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REFERENCES lation coefﬁcients were 0.9990. The recovery of methylephedrine from urine was 101%. Selected drugs were in- 1. Wu J, Lord H, Pawliszyn J, Determination of stimulants in vestigated for potential interference and for drugs elut- human urine and hair samples by polypyrrole coated capil- ing in the monitored time window; their retention times lary in-tube solid phase microextraction coupled with liquid are given in minutes in parentheses: acebutolol, al- chromatography–electrospray mass spectrometry, Talanta 54: prenolol, amphetamine (14.94), atenolol (11.71), betaxolol, 655–672 (2001). bisoprolol, bunolol, caffeine (22.6), dimetamphetamine, 2. Stanaszek R, Piekoszewski W, Simultaneous determina- ethylephedrine, labetalol, mepindolol (18.64), metham- tion of eight underivatized amphetamines in hair by high- phetamine (18.5), metoprolol, morphine (8.05), nadolol, performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS), octapamine, oxprenolol, penbutolol, phentermine, pin- J Anal Toxicol 28: 77–85 (2004). dolol, propranolol, salbutamol (10.45), sotalol (11.02), synephrine, terbutaline (10.01), timolol, and tyramine.

METHYLEPHEDRINE HYDROCHLORIDE REFERENCE

CHEMISTRY 1. Gmeiner G, Geisendorfer T, Kainzbauer J, et al., Quantiﬁcation of ephedrines in urine by column-switching high-performance Methylephedrine is a sympathomimetic agent. Its liquid chromatography, J Chromatogr B 768: 215–221 (2002). chemical name is (1RS,2RS)-2-dimethylamino-1- phenylpropan-1-ol hydrochloride. Its molecular formula is C11H17NO·HCl, with a molecular weight of 215.7 and a CAS number of 18760-80-0. METOCLOPRAMIDE HYDROCHLORIDE

CHEMISTRY METHOD Metoclopramide hydrochloride is an antimetic. Its chem- Gmeiner et al. [1] described a column-switching Assay 1 ical name is 4-amino-5-chloro-N-(2-diethylaminoethyl)-2- HPLC method for the determination of five ephedrines on methoxybenzamide hydrochloride monohydrate. Its molec- the International Olympic Committee list of prohibited ular formula is C H ClN O ·HCl·H O, with a molecular substances, norephedrine, norpseudoephedrine (cathine), 14 22 3 2 2 weight of 354.3 and a CAS number of 54143-57-6. Metoclo- ephedrine, pseudoephedrine, and methylephedrine. A pramide hydrochloride is a white or practically white, odor- ThermoQuest liquid chromatograph consisting of a model less or practically odorless, crystalline powder. Metoclo- P4000 quaternary pump (pump 1), model AS3000 au- pramide hydrochloride is very soluble in water and freely tosampler, model UV6000L diode-array detector, six-port soluble in alcohol. It is sparingly soluble in chloroform and valve, and a Beckman model 112 solvent delivery module practically insoluble in ether. The drug has pK values (pump 2) was used. The stationary phase was a laboratory- a of 0.6 and 9.3. Metoclopramide hydrochloride should be made Hypersil BDS C analytical column (150 × 3 mm, 18 stored in airtight containers and protected from light. 3 ␮m particle size). The column temperature was maintained at 35◦C. The precolumn was a laboratory-made Hy- persil ODS C18 column (20 × 3 mm, 3 ␮m particle size). METHOD The mobile phase was 3% acetonitrile in 0.1% sulfuric acid aqueous solution and was delivered at 1 mL/min. After Assay 1 Liang et al. [1] described the determination of injection of a sample, the precolumn was washed with metoclopramide in human plasma using HPLC. A Perkin- water by pump 2 at 1 mL/min for 5 min and then the Elmer liquid chromatograph equipped with a model six-port valve was switched so that the analytes retained LC235C UV detector and model 1022LC integrator was on precolumn were backflushed to the analytical column used. The stationary phase was a Waters stainless-steel and separated. UV detection was performed at 205 and Nova-Pak C18 column (150 × 3.9 mm, 5 ␮m particle size). 214 nm. The column temperature was maintained at 45◦C. The mo- A stock solution of etilefrine and bamethan at 1 mg/mL bile phase consisted of methanol and 0.05 M monobasic each was prepared in methanol and used as internal stan- ammonium phosphate buffer (pH 3.1) (50 : 50) and was dards. Standards were prepared by spiking drug-free urine delivered at 1 mL/min. The phosphate buffer was prepared with ephedrines. An aliquot of 1 mL of urine or stan- by dissolving 5.75 g of monobasic ammonium phosphate in dard was spiked with 10 ␮L of the internal standard so- water, mixing with 15 mL of glacial acetic acid, and filling lution and assayed directly. Under these conditions, reten- to 1000 mL. UV detection was performed at 310 nm. The tion times for etilefrine, norephedrine, cathine, ephedrine, injection volume was 40 ␮L. pseudoephedrine, methylephedrine, and bamethan were A stock solution of metoclopramide at 1.0 mg/mL was 8.29, 10.53, 11.40, 13.45, 14.43, 15.56, and 18.87 min, re- prepared and stored at 4◦C. Standards were prepared by spectively. Etilefrine and bamethan were used as retention spiking blank human plasma with this stock solution. An markers only. aliquot of 0.5 mL of plasma sample or standard was mixed Calibration curves for methylephedrine were con- with 4 mL of acetonitrile, vortexed for 1 min, and cen- structed over the range from 2.48 to 49.7 ␮g/mL. Corre- trifuged at 3000 rpm for 10 min. The supernatant was P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

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separated, evaporated to dryness at 40◦C under a gentle monobasic sodium phosphate buffer (pH 4.1), mixed with stream of nitrogen, reconstituted with 200 ␮L of mobile 4 mL of ethyl acetate, vortexed for 2 min, and centrifuged phase, and assayed. Under these conditions, the retention at 1500 g for 5 min. The organic layer was separated, time of metoclopramide was about 3.1 min (estimated from mixed with 5 mL of 0.1 M dibasic sodium phosphate buffer the published chromatogram). (pH 7.5), vortexed for 2 min, and centrifuged. The organic Calibration curves for metoclopramide were con- phase was collected, evaporated to dryness at 60◦C under structed over the range from 250 to 10000 ng/mL. Corre- nitrogen, reconstituted with 100 ␮L of 35% acetonitrile lation coefficients were 0.9988. Recovery of the drug from in 0.01 M phosphate buffer (pH 3.0), and assayed. Under plasma ranged from 98.1 and 102.4%. Intraday and in- these conditions, retention times for chlorothiazide, hy- terday coefficients of variation were less than 1.97% and drochlorothiazide, quinethazone, chlorthalidone, methy- 1.65%, respectively. The limit of detection was 10 ng/mL. clothiazide, frusemide, metolazone, mefruside, bendroflu- There was no interference from endogenous materials in azide, cyclopenthiazide, and bumetanide were about 1.9, plasma. 2.2, 3.4, 4.2, 4.7, 5.1, 5.3, 6.2, 6.8, 7.2, and 9.3 min, respectively (estimated from the published chromatogram). Clopamide had the same retention time as did methycloth- REFERENCE iazide. These two drugs were identified by comparing UV spectra of the peak between 220 and 400 nm. 1. Liang Y, Guo J, Wu D, High-performance liquid chromatographic assay of metoclopramide in human plasma, Bull Acad The mean recovery of metolazone from urine was 80%. Mil Med Sci (China) 19: 300–302 (1995). There was no interference with this screening procedure from the following drugs: acetylsalicylic acid, allopurinol, alprenolol, atenolol, captopril, carbimazole, clonidine, coloxyl, danthron, diazepam, digoxin, doxepin, gliben- METOLAZONE clamide, hydralazine, indomethacin, labetalol, metformin, methyldopa, metoprolol, mianserin, minoxidil, nifedip- CHEMISTRY ine, nitrazepam, oxazepam, oxprenolol, pindolol, prazosin, proparanolol, salbutamol, senokot, theophylline, and tri- Metolazone is a quinazoline-derivative diuretic. Its chem- fluoperazine. ical name is 7-chloro-1,2,3,4-tetrahydro-2-methyl-4-oxo-3- o-tolylquinazoline-6-sulfonamide. Its molecular formula is C16H16ClN3O3S, with a molecular weight of 365.8 and a REFERENCE CAS number of 17560-51-9. Metolazone is a white powder. It is practically insoluble in water and sparingly soluble in 1. Fullinfaw RO, Bury RW, Moulds RFW, Liquid chromato- alcohol. The drug has a pKa of 9.7. Metolazone should be graphic screening of diuretics in urine, J Chromatogr 415: stored in airtight containers and protected from light. 347–356 (1987).

METHOD METOPROLOL Assay 1 Fullinfaw et at. [1] described an HPLC CHEMISTRY method for screening chlorothiazide, hydrochlorothiazide, quinethazone, chlorthalidone, methyclothiazide, Metoprolol is a cardioselective ␤-blocker. Its chem- clopamide, frusemide, metolazone, mefruside, bendroflu- ical name is (±)-1-isopropylamino-3-[4-(2-methoxy- azide, cyclopenthiazide, and bumetanide in human urine. ethyl)phenoxy]propan-2-ol. Its molecular formula is A Hewlett-Packard 1090A liquid chromatograph equipped C H NO , with a molecular weight of 267.4 and a CAS with a photodiode array detector was used. The stationary 15 25 3 number of 54163-88-1. phase was a Merck LiChrosorb RP18 column (125 × 4 mm, 5 ␮m particle size). The column temperature was maintained at 50◦C. The mobile phase consisted of acetonitrile METHODS and 0.01 M phosphate buffer (pH 3.0) and was delivered at 1.5 mL/min in a gradient mode. The acetonitrile content of Assay 1 Li et al. [1] developed an LC-MS/MS method mobile phase was increased from 10% at 1.5 min to 35% at for simultaneous determination of 10 antiarrhythmic 3.5 min. The total runtime was 10 min. UV detection was drugs—diltiazem, amiodarone, mexiletine, propranolol, performed at 271 nm. The injection volume was 5 ␮L. sotalol, verapamil, bisoprolol, metoprolol, atenolol, and Stock solutions of chlorothiazide, hydrochloroth- carvedilol—in human plasma. A Shimadzu LC20AD liq- iazide, quinethazone, chlorthalidone, methyclothiazide, uid chromatographic system equipped with two pumps, a clopamide, frusemide, metolazone, mefruside, bendroflu- vacuum degasser, and an autosampler was employed. The azide, cyclopenthiazide, and bumetanide at 1 mg/mL were stationary phase was a Capcell Pak C18 column (50 × 2.0 separately prepared in ethanol and stored at 4◦C. Working mm, 5 ␮m particle size). Solvent A was 0.02% formic acid solutions of these drugs were prepared by diluting stock in acetonitrile, and solvent B 0.02% was formic acid in wa- solutions with ethanol. Standards were prepared by spik- ter. The mobile phase was delivered in a gradient mode ing blank human urine with working solutions. An aliquot from 95% B to 50% B in the first 3.5 min, then from 50% of 2 mL of urine samples was acidified with 2 mL of 1 M B to 5% B in the next 0.5 min, and returned to 95% B P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

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in 0.5 min followed by 3-min equilibration. The flow rate S-(−)-Methylchloroformate solution (1%) was prepared was 0.3 mL/min. The temperature of the autosamper was in acetonitrile that was previously dried with anhydrous maintained at 4◦C. sodium sulfate. Stock solutions of (±)-metoprolol (0.1 and An ABI-SCIEX API3000 triple quadrupole tandem 1 mg/mL), (−)-metoprolol (1 mg/mL), and (±)-toliprolol (in- mass spectrometer with a TurboIonSpray source was cou- ternal standard, 1 mg/mL) were prepared in methanol and pled to the liquid chromatograph as a detector. It was op- stored at −20◦C in the dark. A urine sample (2.0 mL) erated in positive ionization mode. The ionspray voltage was spiked with 30 ␮g of internal standard, alkalinized was set at 2.5 kV, source temperature at 450◦C, collision- to pH 12 with 1.0 mL of 2 M potassium carbonate solution, activated dissociation at 12, and the collision gas was ni- mixed with 1.0 g of sodium chloride, and extracted twice trogen. The declustering potential was 33 V and collision with 5 mL of ethyl acetate. The organic layer was col- energy 27 V. Analytes were monitored in multiple-reaction lected, dried with sodium sulfate, evaporated to dryness monitoring (MRM) mode: m/z 268.5 → 116.2, 279.2 → at 50◦C under a stream of nitrogen, mixed with 100 ␮L 124.2, 281.2 → 156.2, and 311.2 → 156.2 for metoprolol, of 0.4% triethylamine solution and 100 ␮Lof1%S-(−)- sulfisomedine, sulfamethoxydiazine, and sulfadimethox- methylchloroformate solution, incubated at room tempera- ine, respectively. ture for 1 h, evaporated to dryness under a stream of nitro- Sulfamethoxydiazine, sulfadimethoxine, and sulfisome- gen, reconstituted with 300 ␮L of methanol, and assayed. dine as internal standards 200 ng/mL were prepared in Retention times for (−)-methylchloroformate derivatives acetonitrile. Stock solutions of drugs were prepared in of (−)-metoprolol, (+)-metoprolol, (−)-toliprolol, and (+)- methanol. Working solutions were prepared by diluting toliprolol were 19.7, 20.6, 23.3, and 24.5 min, respectively. stock solutions in methanol/water (1 : 1) and were stored Calibration curves for (+)-metoprolol and (−)- at −20◦C. Standards were prepared by spiking blank hu- metoprolol were constructed in the concentration range man plasma with working solutions. Plasma samples, of 0.75–22.5 ␮g/mL. Correlation coefficients were greater standards, and controls (100 ␮L each) were mixed with 200 than 0.997. The recovery of (+)-metoprolol from urine ␮L of internal standard, vortexed for 10 s, and centrifuged ranged from 86.3% to 90.5%; the recovery of (−)-metoprolol at 16,000 g for 3 min. An aliquot (50 ␮L) of the supernatant from urine ranged from 86.6% to 89.7%. Intraassay and was mixed with 150 ␮L of water and assayed. The injec- interassay coefficients of variation for (+)-metoprolol and tion volume was 5 ␮L. Under these conditions, retention (−)-metoprolol were less than 9.7%. The limit of detection times of metoprolol, sulfisomedine, sulfamethoxydiazine, for each enantiomer of metoprolol was 5 ng. and sulfadimethoxine were 3.2, 2.8, 3.9, and 4.8 min, respectively. A calibration curve for metoprolol was constructed in Assay 3 Umezawa et al. [3] described the simultaneous the range from 5 to 1000 ng/mL. The correlation coefficient determination of four ß-blockers—acebutolol, labetalol, was 0.9992. The accuracy ranged from 96.4% to 106.2%. metoprolol, and propranolol—in human plasma using LC- The recovery from plasma ranged from 85.0% to 90.6%. MS/MS. An Agilent 1100 Series system consisting of a Intraday and interday coefficients of variation were less model G1315A diode-array detector, model G1313A au- than 7.9% and 12.0%, respectively. tosampler, and model G1322A vacuum membrane degasser was utilized. The stationary phase was a Shodex MSpak GF-310 4B column (50 × 4.6 mm, 6 ␮m particle Assay 2 Li et al. [2] described the determination of size) protected by a 2-␮m SUMIPAX PG-ODS inline filter. the enantiomers of metoprolol in human urine by high- Solvent A was 10 mM ammonium acetate aqueous solu- performance liquid chromatography with fluorescence de- tion, and solvent B acetonitrile. The mobile phase was detection. A Hewlett-Packard (HP) 1090 liquid chromato- livered at 0.55 mL/min in a gradient mode at 100% A from graphic system equipped with an ABI Analytical Spec- 0 to 3 min, decreased to 0% A in 1 min, and stayed at 0% A troflow 980 fluorescence detector and an HP G1307A from 4 to 9.5 min. The column was reequilibrated at 100% ChemStation data system was used. The stationary phase Afor5.5min. was a Phenomenex Hypersil 5 C18 column (250 × 4.6 mm) An Applied Biosystems/MDS SCIEX API2000 triple- protected by a laboratory-packed HP ODS Hypersil pre- quadrupole mass spectrometer equipped with a TurboIon- column (20 × 4.0 mm, 30 ␮m particle size). The mobile Spray ion source was operated in the positive mode: phase consisted of 0.1 M phosphate buffer and methanol. TurboIonSpray temperature 490◦C; ion-source voltage 3 The phosphate buffer was prepared by dissolving 13.8 g of kV; ring voltage 390 V; nebulizer gas (high-purity air) 20 monobasic sodium phosphate monohydrate and 1.59 g of psi; heater gas (high-purity air) 80 psi; curtain gas (high- propylamine hydrochloride in 1 L of water and adjusting purity nitrogen) 40 psi; orifice voltages 41 V for acebutolol, the pH to 3.2 with concentrated phosphoric acid. The mo- 25 V for labetalol, 40 V for metoprolol, 51 V for propranolol, bile phase was delivered at 1.0 mL/min in a gradient mode and 40 V for pindolol; collision gas (nitrogen) 4; and colli- and increased from 75% methanol to 85% methanol from sion energies −29 eV for acebutolol, −22 eV for labetalol, 0 to 15 min, held at 85% methanol from 15 to 20 min, in- −25 eV for metoprolol, −25 eV for propranolol, and −25 creased to 90% methanol at 25 min, and then held at 90% eV for pindolol. Quantification was performed in selective- methanol from 25 to 28 min. The fluorescence detection reaction monitoring (SRM) mode using ion transitions at was carried out at an excitation wavelength of 223 nm and m/z 337 → 116 for acebutolol, m/z 329 → 311 for la- an emission wavelength of 340 nm. The injection volume betalol, m/z 268 → 116 for metoprolol, m/z 260 → 116 for was 10 ␮L. propranolol, and m/z 249 → 116 for pindolol, respectively. 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Stock solutions of acebutolol, labetalol, metoprolol, pro- for norfloxacin, m/z 361.8 → 317.9 for ofloxacin, and m/z pranolol, and pindolol (as internal standard) at 1 mg/mL 359.9 → 315.9 for enrofloxacin, respectively. were separately prepared in methanol. Working solutions Stock solutions of drugs were prepared in methanol, were prepared by diluting stock solutions with 10 mM except that antibiotics were prepared in a mixture of ammonium acetate in water. Calibrators were prepared methanol and 0.01 M hydrochloric acid (1 : 1, vol/vol) and by spiking drug-free plasma with working solution. An stored at −18◦C. Working solutions were prepared daily by aliquot of 1 mL of a plasma sample or calibrator was mixed diluting stock solutions with the same solvents. Standards with 3 mL of 13.3 mM ammonium acetate aqueous solution were prepared by spiking noncontaminated groundwater and centrifuged at 9000 rpm for 10 min. The supernatant with working solutions and internal standards. A sample was filtered through a 0.2-␮m Whatman GD/X syringe fil- (100, 250, 500, and 1000 mL for sewage influent, sewage ter (13 mm) and assayed. The injection volume was 100 effluent, surface water, and groundwater, respectively) was ␮L. Under these conditions, retention times of labetalol, adjusted to pH 10.0 with 2 M sodium hydroxide solution, metoprolol, acebutolol, propranolol, and pindolol were 6.6, spiked with 500 ng of the internal standards, and filtered 6.9, 7.2, 7.8, and 7.9 min, respectively. through a 0.45-␮m Schleicher & Schuell GF 6 filter that Calibration curves for metoprolol were constructed in was previously washed with n-hexane, acetone, methanol, the range from 10 to 1000 ng/mL. Correlation coefficients and water. It was then loaded onto a Waters Oasis HLB were greater than 0.9991. The effect of ion suppression on solid-phase extraction cartridge (3 mL, 60 mg) by means of the analysis of metoprolol was less than 23.0%. The re- PTFE tubes at flow rates of 2, 5, 10, and 20 mL/min (sewage covery of metoprolol from plasma ranged from 73.5% to influent water, sewage effluent water, surface water, and 83.4%. The accuracy ranged from 94.5% to 120%. Intra- groundwater, respectively), which was pre-conditioned se- day and interday coefficients of variation were less than quentially with 2 mL of n-hexane,2mLofacetone,10mL 4.7% and 10.9%, respectively. The limit of detection was of methanol, and 10 mL of noncontaminated groundwater 1 ng/mL. (pH adjusted to 10.0); pulled through the cartridge; washed with 2 mL of 5% methanol in 2% aqueous ammonium hy- Assay 4 Vieno et al. [4] developed an LC-MS/MS method droxide; dried with a stream of nitrogen for 30 min; eluted for the detection of acebutolol, atenolol, metoprolol, sotalol, with 4 × 1 mL of methanol; evaporated to near dryness carbamazepine, ciprofloxacin, ofloxacin, and norfloxacin in under a stream of nitrogen, reconstituted with 20 ␮Lof drinking water, surface water, and sewage treatment plant methanol and 480 ␮L of 1% acetic acid; and assayed. Under water. An Agilent 1100 Series system consisting of a binary these conditions, retention times of sotalol, atenolol, nor- pump, vacuum degasser, autosampler, and a thermostated floxacin, ofloxacin, ciprofloxacin, enrofloxacin, acebutolol, column oven was used. The stationary phase was an Agi- metoprolol, alprenolol, carbamazepine, and dihydrocarba- lent Zorbax XDB C18 column (50 × 2.1 mm, 5 ␮m particle mazepine were 3.3, 4.4, 9.8, 9.8, 10.2, 10.9, 11.1, 11.2, 15.4, size) protected by an Agilent narrowbore guard column 17.6, and 17.8 min, respectively. (12.5 × 2.1 mm, 5 ␮m particle size). The column tempera- Calibration curves for metoprolol were constructed in ture was maintained at 30◦C. The mobile phase consisted the range from 0.40 to 6000 ␮g/L. Correlation coefficients of acetonitrile and 1% acetic acid in water and was deliv- were greater than 0.995. Average recoveries of metoprolol ered at 250 ␮L/min in a gradient mode from 3% to 28% from groundwater and tapwater, surface water, sewage ef- acetonitrile in 12 min and to 53% acetonitrile in another fluent water, and sewage influent water were 90%, 104%, 5 min, kept at 53% acetonitrile for 1 min, and then re- 87%, and 93%, respectively. Limits of quantification in turned to the initial condition in 1 min. The column was drinking water, surface water, sewage effluent water, and equilibrated at 3% acetonitrile for 8 min. sewage influent water were 2.2, 3.8, 9.1, and 21 ng/L, re- A Micromass Quattro Micro triple quadrupole mass spectively. spectrometer equipped with an electrospray ionization in- This LC-MS/MS method was successfully applied for terface was operated in positive-ion mode: desolvation the determination of metoprolol in sewage influent water, gas 640 L/h, nebulizing gas 30 L/h, collision gas (argon) sewage effluent water, and their recipient rivers. 2.8 × 10−3 mbar, source temperature 120◦C, and desolvation temperature 325◦C. Cone voltage (V) and collision energy (eV) were 28 and 20 for acebutolol, 30 and 23 for Assay 5 Delamoye et al. [5] developed an HPLC method atenololol, 25 and 15 for metoprolol, 30 and 23 for sotalol, for simultaneous determination of 13 ß-blockers and one 25 and 15 for alprenolol (internal standard), 29 and 18 metabolite: atenolol, sotalol, diacetolol, carteolol, nadolol, for carbamazepine, 35 and 21 for dihydrocarbamazepine pindolol, acebutolol, metoprolol, celiprolol, oxprenolol, la- (internal standard), 30 and 17 for ciprofloxacin, 28 and betalol, propranolol, tertatolol, and betaxolol. A Spec- 16 for norfloxacin, 29 and 18 for ofloxacin, and 28 and 18 tra liquid chromatographic system consisting of a model for enrofloxacin (internal standard), respectively. Quan- P1000XR quaternary gradient pump, model AS3000 au- tification was performed in multiple-reaction monitoring toinjector with a 100-␮L loop, and model 6000 LP (MRM) mode using ion transitions at m/z 336.8 → 116.0 photodiode-array detector was used. The stationary phase for acebutolol, m/z 267.0 → 144.9 for atenolol, m/z 267.9 was a ThermoHypersil Hypurity C18 column (250 × 4.6 → 190.9 for metoprolol, m/z 254.8 → 132.9 for sotalol, m/z mm, 5 ␮m particle size) protected by a C18 precolumn (4 249.9 → 172.9 for alprenolol, m/z 237.0 → 193.9 for carba- × 4.4 mm, 5 ␮m particle size). The column temperature mazepine, m/z 239.0 → 193.9 for dihydrocarbamazepine, was maintained at 35◦C. UV detection was performed at m/z 331.9 → 287.9 for ciprofloxacin, m/z 319.8 → 275.9 220 nm. The injection volume was 80 ␮L. P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

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Stock solutions of these compounds at 1.0 g/L each were GF/C filter (90 mm i.d.) with a layer of Celite; acidified prepared in methanol. Working solutions were prepared by to pH 3 with 1 M hydrochloric acid; loaded onto a Waters diluting stock solutions with methanol. A stock solution of Oasis MCX cartridge (6 mL, 150 mg, 30 ␮m) at a flow rate medroxalol at 50.0 mg/L in methanol was used as an in- of 10–15 mL/min, which was preconditioned with 6 mL of ternal standard. These stock and working solutions were methanol followed by 10 mL of water at pH 3; dried for stored at −20◦C. An aliquot of 1 mL of plasma, standard, 10 min under vacuum; washed with 100 mL of water at or control was spiked with 20 ␮L of the internal standard, pH 3 followed by 6 mL of methanol; eluted with 8 mL of a mixed with 500 ␮L of 1 M sodium carbonate (pH 9.7), ex- mixture of dichloromethane, 2-propanol, and ammonium tracted with 7 mL of chloroform–pentanol–diethyl ether hydroxide(78:20:2,vol/vol/vol);evaporatedtodryness (6 : 2: 1, vol/vol/vol), shaken for 15 min, and centrifuged at 40◦C under a stream of nitrogen, reconstituted in 1.0 mL at 3000 g for 5 min. The organic phase was collected, mixed of mobile phase B; filtered through a 0.45-␮m nylon syringe with 250 ␮L of 0.05 M phosphoric acid (pH 2.1), shaken for filter; and assayed. Under these conditions, retention times 10 min, and centrifuged at 3000 g for 5 min. The aque- for acebutolol, alprenolol, atenolol, bisoprolol, labetalol, ous phase was collected and assayed. Under these condi- metoprolol, nadolol, pindolol, propranolol, sotalol, timolol, tions, retention times for atenolol, sotalol, diacetolol, car- clenbuterol, fenoterol, salbutamol, and terbutaline were teolol, nadolol, pindolol, acebutolol, metoprolol, celiprolol, 17.36, 24.32, 8.96, 21.77, 22.11, 18.02, 13.60, 14.15, 23.87, oxprenolol, medroxalol, labetalol, propranolol, tertatolol, 7.85, 17.02, 17.91, 11.50, 6.97, and 6.53 min, respectively. and betaxolol were 5.1, 5.6, 7.9, 9.2, 9.9, 10.5, 14.5, 15.4, Calibration curves for metoprolol were constructed over 18.8, 20.4, 21.2, 21.8, 24.6, 25.1, and 25.9 min, respectively. the range from 50 to 500 pg/␮L. The mean recovery of the Calibration curves for metoprolol were constructed over drug from water ranged from 85% to 91%. The limit of the range from 25 to 1000 ng/mL. The mean correlation detection was 8 ng/L. coefficient was 0.997. The mean accuracy was 101.3% at 100 ng/mL. The mean recovery was 100%. Intraday and interday coefficients of variation were 8.31% and 11.80%, REFERENCES respectively. Limits of detection and quantification were 7 and 25 ng/mL, respectively. 1. Li S, Liu G, Jia J, et al., Simultaneous determination of ten an- tiarrhythic drugs and a metabolite in human plasma by liquid chromatography–tandem mass spectrometry, J Chromatogr B Lee et al. [6] developed an LC-MS/MS method Assay 6 847: 174–181 (2007). for the simultaneous determination of 12 ß-blockers and 2. Li F, Cooper SF, Cote M, Determination of the enantiomers ß -agonists in sewage samples. A Waters 2695 separation 2 of metoprolol and its major acidic metabolite in human urine module was used. The stationary phase was an Agilent by high-performance liquid chromatography with fluorescence × ␮ Zorbax SB C8 column (150 2.1 mm, 3.5 m particle size) detection, J Chromatogr B 668: 67–75 (1995). × ␮ protected by a SB C8 guard column (12.5 2.1 mm, 5 m 3. Umezawa H, Lee X-P, Arima Y, et al., Simultaneous de- particle size). The column temperature was maintained at termination of ß-blockers in human plasma using liquid ◦ 35 C. Mobile phase A was a mixture of water, acetonitrile, chromatography-tandem mass spectrometry, Biomed Chro- and formic acid (94.5 : 5.0 : 0.5, vol/vol/vol), and mobile matogr 22: 702–711 (2008). phase B was a mixture of acetonitrile and formic acid (99.5 4. Vieno NM, Tuhkanen T, Kronberg L, Analysis of neutral : 0.5, vol/vol). The mobile phase was delivered in a gradient and basic pharmaceuticals in sewage treatment plants and mode from 100% A to 75% A in 13 min, held at 75% A for in recipient rivers using solid phase extraction and liq- 13 min, and then pumped at 100% A for another 14 min. uid chromatography–tandem mass spectrometry detection, The flow rate was 0.2 mL/min. The injection volume was J Chromatogr A 1134: 101–111 (2006). 10 ␮L. The total runtime of an injection was 40 min. 5. Delamoye M, Duverneuil C, Paraire F, et al., Simultaneous de- A Micromass Quattro Ultima triple quadrupole mass termination of thirteen ß-blockers and one metabolite by gradi- spectrometer equipped with an electrospray ionization ent high-performance liquid chromatography with photodiode- source was operated in the positive ion mode. The major array UV detection, Forensic Sci Intnatl 141: 23–31 (2004). parameters were set as follows: nebulizer gas (nitrogen) 50 6. Lee H-B, Srafin K, Peart TE, Determination of ß-blockers and L/h, desolvation gas (nitrogen) 500 L/h, source tempera- ß2-agonists in sewage by solid-phase extraction and liquid chromatography–tandem mass spectrometry, J Chromatogr A ture 120◦C, desolvation temperature 350◦C, capillary volt- 1148: 158–167 (2007). age 3.45 kV, cone energy 60 V, and collision energy 20 kV. Metoprolol was analyzed in the multiple-reaction monitoring (MRM) mode at ion transitions m/z 268 → 116 for quantitation and m/z 268 → 133 for confirmation. METRONIDAZOLE Individual stock solutions of acebutolol, alprenolol, atenolol, bisoprolol, clenbuterol, fenoterol, labetalol, meto- CHEMISTRY prolol, nadolol, pindolol, propranolol, sotalol, terbutaline, and timolol at 1000 ␮g/mL were prepared in acetonitrile Metronidazole is an antiprotozoal. Its chemical name is or methanol. A stock solution of salbutamol was prepared 2-(2-methyl-5-nitroimidazol-1-yl)ethanol. Other names in- at 500 ␮g/mL. These stock solutions were stored at −20◦C. clude Bayer-5360, metronidazol, Metrocream, Vaginyl, and Working solutions were prepared by mixing and diluting Vandazole. Its molecular formula is C6H9N3O3,witha these stock solutions with mobile phase B. An aliquot of molecular weight of 171.2 and a CAS number of 443-48- 250 mL of sewage sample was filtered through a 1.2-␮m 1. Metronidazole occurs as white to pale yellow, odorless P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

322 MEXILETINE HYDROCHLORIDE

crystals or crystalline powder. Metronidazole is sparingly drugs—diltiazem, amiodarone, mexiletine, propranolol, soluble in water and alcohol and slightly soluble in chlo- sotalol, verapamil, bisoprolol, metoprolol, atenolol, and roform and ether. Metronidazole should be stored at 25◦C carvedilol—in human plasma. A Shimadzu LC20AD liq- and protected from light. uid chromatographic system equipped with two pumps, a vacuum degasser, and an autosampler was employed. The stationary phase was a Capcell Pak C18 column (50 × 2.0 METHOD mm, 5 ␮m particle size). Solvent A was 0.02% formic acid in acetonitrile, and solvent B was 0.02% formic acid in wa- Assay 1 Holt et al. [1] described an HPLC assay for the si- ter. The mobile phase was delivered in a gradient mode multaneous determination of chloramphenicol, metronida- from 95% B to 50% B in the first 3.5 min, then from 50% zole, cefuroxime, cephalexin, ceftazidime, ampicillin, and B to 5% B in the next 0.5 min, and returned to 95% B in benzylpenicillin. A liquid chromatographic system com- 0.5 min followed by 3-min equilibration. The flow rate was prising a Waters model 510 pump, model 481 variable- 0.3 mL/min. The temperature of autosamper was main- wavelength UV detector, and a Rheodyne injector with tained at 4◦C. ␮ a 20- L loop was used. The stationary phase was a An ABI-SCIEX API3000 triple quadrupole tandem × LiChrosorb C18 stainless-steel column (250 4.6 mm, mass spectrometer with TurboIonSpray source was cou- ␮ 10 m particle size). The mobile phase was 15% methanol pled to the liquid chromatograph as a detector. It was op- in 100 mM sodium phosphate buffer (pH 6.0). The flow rate erated in positive ionization mode. The ionspray voltage was 1.7 mL/min. UV detection was carried out at 214 and was set at 2.5 kV, source temperature at 450◦C, collision- 320 nm. activated dissociation at 12, and the collision gas nitrogen. ␮ Proteins in serum samples (100 L each) were pre- The declustering potential was 20 V and collision energy 22 cipitated with 2 volumes of 4% perchloric acid contain- V. Analytes were monitored in multiple-reaction monitoring tinidazole 25 mg/L as an internal standard and cen- ing mode (MRM): m/z 180.2 → 58.1, 279.2 → 124.2, 281.2 trifuged. Supernatants were assayed immediately. The → 156.2, and 311.2 → 156.2 for mexiletine, sulfisomedine, ␮ injection volume was 20 L. Under these conditions, re- sulfamethoxydiazine, and sulfadimethoxine, respectively. tention times of metronidazole and tinidazole were 5.0 and Sulfamethoxydiazine, sulfadimethoxine, and sulfisome- 7.8 min, respectively. Metabolites of metronidazole did not dine as internal standards 200 ng/mL were prepared in interfere with its analysis. acetonitrile. Stock solutions of drugs were prepared in Linear calibration curves for metronidazole were estab- methanol. Working solutions were prepared by diluting lished in the range of 1–100 mg/L, with correlation coeffi- stock solutions in methanol/water (1 : 1) and were stored cients of 1.00. The coefficient of variation of the assay was at −20◦C. Standards were prepared by spiking blank hu- 2.0%. The interday coefficient of variation was less than man plasma with working solutions. Plasma samples, 5%. The mean recovery of metronidazole from serum was standards, and controls (100 ␮L each) were mixed with 93%. The limit of detection was 1.0 mg/L. 200 ␮L of internal standard, vortexed for 10 s, and centrifuged at 16,000 g for 3 min. An aliquot (50 ␮L) of the ␮ REFERENCE supernatant was mixed with 150 L of water and assayed. The injection volume was 5 ␮L. Under these con- 1. Holt DE, de Louvois J, Hurley R, et al., A high performance ditions, retention times of mexiletine, sulfisomedine, sul- liquid chromatography system for the simultaneous assay of famethoxydiazine, and sulfadimethoxine were 3.4, 2.8, 3.9, some antibiotics commonly found in combination in clinical and 4.8 min, respectively. samples, J Antimicrob Chemother 26: 107–115 (1990). A calibration curve for mexiletine was constructed in the range from 10 to 2000 ng/mL. The correlation coefficient was 0.9989. The accuracy ranged from 95.3% to MEXILETINE HYDROCHLORIDE 103.7%. The recovery from plasma ranged from 90.2% to 97.1%. Intraday and interday coefficients of variation were less than 7.2% and 6.5%, respectively. CHEMISTRY

Mexiletine hydrochloride is an antiarrhythmic agent. Its chemical name is 1-methyl-2-(2,6-xylyloxy)ethylamine hy- Assay 2 Dasgupta et al. [2] reported a gas chromatography–electron ionization and chemical ionization drochloride. Its molecular formula is C11H17NO·HCl, with a molecular weight of 215.7 and a CAS number of 5370- mass spectrometric analysis of serum mexiletine 01-4. Mexiletine hydrochloride is a white or almost white, concentration after derivatization with 2,2,2-trichlo- odorless or almost odorless, crystalline powder. It is freely roethylchloroformate. For the gas chromatography– soluble in water and in methanol, sparingly soluble in chlo- electron ionization mass spectrometric analysis, a roform, and practically insoluble in ether. Hewlett-Packard model 5890 gas chromatograph was coupled with an HP model 5970 mass selective detector. For the gas chromatography–chemical ionization mass spec- METHODS trometric analysis, a Hewlett-Packard model 5890 Series II gas chromatograph was coupled to a Hewlett-Packard Assay 1 Li et al. [1] developed an LC-MS/MS method model 5972 mass selective detector. Methane was used as for simultaneous determination of 10 antiarrhythmic the ionizing gas. P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

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The stationary phase was an Ultra-1 column (25 m × volume, and assayed. The injection volume was 2–3 ␮L. 0.2 mm) that was crosslinked with methyl silicone gum at Under these conditions, retention times for the derivatized a film thickness of 0.33 ␮m. The oven temperature was mexiletine and the derivatized N-propylamphetamine (in- programmed at the initial temperature of 195◦Cfor5min, ternal standard) were 8.6 and 8.1 min, respectively. then increased to 300◦Cat20◦C/min, and kept at 300◦C A calibration curve for mexiletine was linear in the for 3 min. The total runtime was 13.25 min. The injection range of 0.2–4 mg/L. Within-run and between-run co- port temperature was 250◦C. A range of m/z 40–400 was efficients of variation at 1 mg/L level were 1.9% and scanned for electron ionization mass spectra, and a range 2.5%, respectively. The limit of detection was 0.1 mg/L. of m/z 50–400 was scanned for chemical ionization mass This assay was carryover-free. The regression relation- spectra, respectively. Ions were monitored at m/z 56, 58, ship of this assay with an established GC method with 91, 102, 122, 131, 232, 234, 260, and 262. nitrogen–phosphorus detection was y = 0.92x + 0.09 (r = Mexiletine stock solution (1 mg/mL) was prepared 0.95). in dichloromethane, and N-propylamphetamine (internal standard, 0.1 mg/mL) was prepared in methanol. A serum sample (1 mL) was supplemented with 10 ␮Lofthe REFERENCES internal standard solution, mixed with 1 mL of borate 1. Li S, Liu G, Jia J, et al., Simultaneous determination of ten an- buffer (pH 9.8) that was prepared by dissolving 20 g tiarrhythic drugs and a metabolite in human plasma by liquid sodium tetraborate decahydrate in 1 L of deionized wa- chromatography–tandem mass spectrometry, J Chromatogr B ter, extracted with 10 mL of dichloromethane by rotat- 847: 174–181 (2007). ing for 15 min, and centrifuged at 1500 g for 5 min. The 2. Dasgupta A, Appenzeller P, Moore, J, Gas chromato- lower organic layer was collected, evaporated under air graphy–electron ionization and chemical ionization mass spec- until 50–100 ␮L of the solution, mixed with 50 ␮Lof trometric analysis of serum mexiletine concentration after 2,2,2-trichloroethylchloroformate (derivatizing agent), in- derivatization with 2,2,2-trichloroethyl chloroformate: a novel cubated at 70◦C for 30 min, and further concentrated. The derivative, Ther Drug Monit 20: 313–318 (1998). injection volume was 2–3 ␮L. Under these conditions, re- 3. Dasgupta A, Yousef O, Gas chromatographic–mass spectro- tention times for the derivatized mexiletine and the deriva- metric determination of serum mexiletine concentration after tized internal standard were 9.6 and 10.6 min, respectively. derivatization with perfluorooctanoyl chloride, a new deriva- A calibration curve for mexiletine was linear in the con- tive, J Chromatogr B 705: 283–288 (1998). centration range of 0.2–2.5 mg/L. Within-run and between- run coefficients of variation at the 1 mg/L concentration level were 1.7% and 3.3%, respectively. The limit of de- MIANSERIN HYDROCHLORIDE tection was 0.1 mg/L. The regression relationship of this assay to an established GC-MS assay without derivatiza- CHEMISTRY tion was y = 0.91x + 0.036 (r = 0.99). This assay was free of carryover and interference from digoxin, digitoxin, Mianserin hydrochloride is a tricyclic antidepres- pheytoin, valproic acid, phenobarbital, diazepam, carba- sant. Its chemical name is 1,2,3,4,10,14b-hexahydro-2- mazepine, doxepin, nordoxepin, amitriptyline, nortripty- methyldibenzo[c,f ]pyrazino[1,2-a]azepine hydrochloride. line, imipramine, desipramine, and protriptyline. Its molecular formula is C18H20N2·HCl, with a molecular weight of 300.8 and a CAS number of 21535-47-7. Mi- Assay 3 Dasgupta and Yousef [3] developed and validated anserin hydrochloride occurs as a white or almost white a GC-MS method for the determination of serum mexile- crystalline powder or as crystals. It is sparingly solu- tine concentration after derivatization with perfluorooc- ble in water, slightly soluble in alcohol, and soluble in tanoyl chloride. GC-MS instruments used were the same dichloromethane. as described in Assay 2 above. The oven temperature was programmed at the initial temperature of 175◦Cfor5min, then increased to 300◦Cat20◦C/min, and kept at 300◦C METHODS for 2 min. The total runtime was 13.25 min. The injection port temperature was 250◦C. Helium was used as a carrier Assay 1 Volin [1] described a GC method for the gas at a column flow rate of 0.29 mL/min and a linear ve- routine determination of tricyclic antidepressants in locity of 21 cm/s. A range of m/z 40 to 600 was scanned for human plasma with a specific nitrogen detector. A electron ionization mass spectra, and a range of m/z 50 Hewlett-Packard model HP5710A gas chromatograph was to 600 was scanned for chemical ionization mass spectra, equipped with a model 18765A nitrogen–phosphorus de- respectively. Ions were monitored at m/z 91, 118, 122, 440, tector and model HP3390A data processor. The stationary 454, 482, and 575. phase was a Supelco coiled glass column (1.8 m × 2mm The serum sample preparation was the same as de- i.d.) containing GP 3% SP2250 on 80/100-mesh Supelco- scribed in Assay 2 above except that the lower organic layer port. Temperatures for column, detector, and injector were in the extraction step was evaporated to dryness under setat240◦C (or 250◦C), 300◦C, and 250◦C, respectively. nitrogen, mixed with 50 ␮L of perfluorooctanoyl chloride The flow rate of the nitrogen carrier gas was 40 mL/min. (derivatizing agent), incubated at 80◦C for 20 min, evapo- The injection volume was 2–5 ␮L. rated again to dryness, reconstituted with 50 ␮Lofethyl Protriptyline at 1.0 mg/mL in methanol was used as an acetate, further concentrated to approximately half of the internal standard. A plasma sample or standard (3 mL) P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

324 MIDAZOLAM HYDROCHLORIDE

was mixed with 1.2 mL of saturated sodium carbonate retention times for melperone and mianserin were 8.8 and (Na2CO3) by shaking vigorously for 2 min, extracted with 29.0 min, respectively. 10 mL of n-hexane/isoamyl alcohol (97 : 3, vol/vol) con- Calibration curves for mianserin were constructed over taining 0.1 ␮g/mL internal standard by shaking for 15 min, the range from 5 to 300 ng/mL. Correlation coefficients and centrifuged at 1000 g for 5 min. A portion (8 mL) were 0.9979. Recovery of the drug from serum ranged of the organic phase was collected, mixed with 1.2 mL from 99.4% to 101.3%. Intraassay and interassay coef- of 0.9 M hydrochloric acid, shaken for 15 min, and cen- ficients of variation were less than 9.4% and 9.7%, re- trifuged. The aqueous phase was collected, mixed vigor- spectively. There was no interference with the assay from ously with 500 mg of anhydrous sodium carbonate for the following drugs and their metabolites (retention times 1min,extractedwith3mLofn-hexane/isoamyl alcohol in minutes): sulpiride (4.1), O-desmethylvenlafaxine (4.8), without internal standard for 15 min, and centrifuged. 9-OH-risperidone (6.6), m-chlorophenylpiperazine (8.0), The organic phase was collected, mixed vigorously with normirtazapine (8.3), zolpidem (10.2), nordoxepin (10.9), 250 mg of anhydrous sodium sulfate, and centrifuged. The diazepam (11.0), benperidol (11.5), normaprotiline (11.5), supernatant was collected, evaporated to dryness at 40◦C dibenzepine (11.5), opipramol (11.6), norfluoxetine (13.4), under a stream of nitrogen, reconstituted in 120 ␮Lof norclozapine (14.4), haloperidol (15.3), norclomipramine methanol, and assayed. Relative retention times to inter- (19.2), trifluperidol (20.8), olanzapine (21.0), ziprasidone nal standard for amitriptyline, trimipramine, imipramine, (26.4), promethazine (28.1), flupenazine (31.0), nefazodone doxepin, nortriptyline, mianserin, iprindole, maprotiline, (32.5), chlorprothixene (36.4), thioridazine (43.2), pimozide and clomipramine were 0.72, 0.74, 0.80, 0.83, 0.86, 0.89, (44.1), carbamazepine, perazine, zotepine, valproate, zopi- 1.04, 1.11, and 1.22 min, respectively. clone, buspirone, lorazepam, and biperidene. A linear calibration curve for mianserin was obtained in the concentration range of 25–175 ␮g/L. The recovery ranged from 86% to 102%. The coefficient of REFERENCES variation was less than 3.9%. There was no interfer- 1. Volin P, Therapeutic monitoring of tricyclic antidepressant ence from perphenazine, nitrazepam, diazepam, levome- drugs in plasma or serum by gas chromatography, Clin Chem promazine, digoxin, atenolol, melperone, chlorpromazine, 27: 1785–1787 (1981). thioridazine, lithium, flunitrazepam, fluphenazine, chlor- 2. Frahnert C. Rao ML, Grasmader K, Analysis of eighteen an- diazepoxide, propranolol, insulin, promazine, and lo- tidepressants, four atypical antipsychotics and active metabo- razepam. lites in serum by liquid chromatography: A simple tool for therapeutic drug monitoring, J Chromatogr B 794: 35–47 (2003). Assay 2 Frahnert et al. [2] reported the analysis of mianserin in human serum by HPLC for therapeutic drug monitoring. A liquid chromatograph consisting of a Bischoff 2200 pump, a Bischoff SDU2003 solvent degasser, MIDAZOLAM HYDROCHLORIDE a Waters WISP 717 autosampler, and a Shimadzu SPD- 10AVP UV detector was used. The stationary phase con- CHEMISTRY sisted of a Macherey–Nagel Nucleosil 100-5-Protect 1 an- × ␮ alytical column (250 4.6 mm, 5 m particle size). The Midazolam is a short-acting benzodiazepine sedative. mobile phase consisted of 25 mM monobasic potassium Its chemical name is 8-chloro-6-(2-fluorophenyl)-1-methyl- phosphate buffer (pH 7.0) and acetonitrile (60 : 40) and 4H-imidazol[1,5-a][1,4]benzodiazepine hydrochloride. Its was isocratically delivered at 1 mL/min. UV detection was molecular formula is C H ClFN ·HCl, with a molecular ␮ 18 13 3 performed at 230 nm. The injection volume was 100 L. weight of 362.2 and a CAS number of 59467-96-8. Mida- A stock solution of mianserin at 1.0 mg/mL was pre- zolam is a white to light yellow, crystalline powder. Mi- pared in methanol. Working solutions were prepared by dazolam has solubilities of approximately 0.24, 1.09, 3.67, diluting this stock solution with water. Standards were 10.3, or > 22 mg/mL in water at pH 6.2, 5.1, 3.8, 3.4, or prepared by spiking blank human serum with working so- 2.8, respectively, at 25◦C. It is freely soluble in ethanol and lutions. Melperone at 3000 ng/mL was used as an internal acetone and soluble in methanol. The drug has a pKa of standard. An aliquot of 1 mL of serum sample or stan- 6.15. dard was centrifuged at 13000 g and at 4◦C for 10 min. An aliquot of 0.9 mL of the supernatant was separated, mixed with 0.1 mL of internal standard and 2.0 mL of 0.1 METHODS M monobasic potassium phosphate buffer (pH 6.0); loaded onto a Varian 3M-Empore extraction disk cartridge (3 mL) Assay 1 Liu et al. [1] reported a capillary GC method for that was preconditioned with 1 mL of methanol followed simultaneous determination of fentanyl and midazolam in by 1 mL of water; pulled through the cartridge; washed human plasma. A Hewlett-Packard 5890 II gas chromato- sequentially with 1 mL of water, 1 mL of 1 M acetic acid, graph equipped with a splitless capillary inlet port and 1mLofn-hexane, 2 mL of n-hexane–ethyl acetate (1 : 1), a nitrogen–phosphorus detector was used. The stationary and 1 mL of methanol; eluted with 1 mL of 2-propanol–25% phase was a Hewlett-Packard crosslinked capillary wide- ammonium solution–dichloromethane (20 : 2: 78); evapo- bore column (methylsiligum, 10 m × 0.53 mm, 2.65 ␮m rated to dryness; reconstituted with 250 ␮L of acetonitrile film thickness). The major parameters were set as follows: and water (3 : 7); and assayed. Under these conditions, column temperature 255◦C, injector temperature 285◦C, P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

MILNACIPRAN HYDROCHLORIDE 325

detector temperature 290◦C, nitrogen carrier gas flow rate sufentanyl at 1 ␮g/mL was prepared daily in water and was 4.4 mL/min, air 105 mL/min, and hydrogen 3.5 mL/min. used as an internal standard. Standards were prepared by The injection volume was 2 ␮L. spiking blank human plasma with working solutions of Stock solutions of midazolam and Ro21-2212 at fentanyl and midazolam. An aliquot of 1 mL of plasma or 1 mg/mL each were prepared in methanol. Stock standard was mixed with 50 ␮L of internal standard, vor- solutions of fentanyl and 1-(2-phenmethyl)-4-N-(N- texed for 10 s, diluted with 2.5 mL of water followed by propionylanilino)piperidine (compound 1) at 1 mg/mL each 0.5 mL of a mixture of 1 M sodium hydroxide aqueous so- were prepared in water. Ro21-2212 was used as an in- lution containing 6% sodium chloride and methanol (80 : ternal standard, and compound 1 was used as a carrier. 20, vol/vol), and shaken vigorously. The SPME procedure Working solutins of 1 ␮g/mL internal standard, 1 ␮g/mL was carried out by exposing a 65-␮m polydimethylsiloxane/ compound 1, 1 ␮g/mL fentanyl, and 10 ␮g/mL midazolam divinylbenzene (PDMS/DVB) fiber in the above mentioned were independently prepared by diluting stock solutions solution for 30 min while being stirred by a magnetic stir- with methanol. Standards were prepared by spiking drug- rer. This PDMS/DVB fiber was washed with water for 30 s free human plasma with working solutions. An aliquot of followed by 20% methanol for another 30 s, dried at room 1mLofplasmaorstandardwasmixedwith0.05mLof temperature in air for 1 min, and injected into the gas compound 1, 0.05 mL of internal standard, and 0.05 mL chromatograph. Under these conditions, retention times of 8 M sodium hydroxide; shaken, extracted with 5 mL of midazolam, fentanyl, and sufentanyl were 16.3, 18.4, of cyclohexane–isopentanol (197 : 3); vortexed; and cen- and 20.0 min, respectively. trifuged at 1350 g for 10 min. The organic phase was sepa- Calibration curves for midazolam were constructed over rated, mixed with 2 mL of 0.125 M sulfuric acid, vortexed the range from 5 to 500 ng/mL. Correlation coefficients for 1 min, and centrifuged. The aqueous phase was col- were 0.9998. The mean accuracy expressed as the rela- lected, alkalinized with 0.07 mL of 8 M sodium hydroxide, tive percentage error was 13.3%. Intraassay and interas- and extracted with 5 mL of n-hexane. The n-hexane phase say coefficients of variation were 2.3% and 7.0%, respec- was evaporated to dryness at 40◦C under a stream of ni- tively. Limits of detection and quantification were 0.4 and trogen, reconstituted with 30 ␮L of ethanol, and assayed. 1 ng/mL, respectively. Under these conditions, retention times of midazolam, fentanyl, and internal standard were 4.7, 6.3, and 7.8 min, respectively. REFERENCES Calibration curves for midazolam were constructed over 1. Liu Y, Wu Y, Zhou J, et al., Capillary GC determination of the range from 5 to 1000 ng/mL. The average correla- fentanyl and midazolam in human plasma, Microchem J 53: tion coefficient was 0.9998. The mean recovery of mida- 130–136 (1996). zolam from plasma was 98.9%. Within-run and between- 2. Dufresne C, Favetta P, Gonin R, et al., Simultaneous deter- run coefficients of variation were 4.4% and 5.2%, respec- mination of fentanyl and midazolam in plasma using direct tively. The limits of detection and quantification were 0.5 solid-phase microextraction before gas chromatography–mass and 5 ng/mL, respectively. There was no interference with spectrometery analysis, Anal Lett 35: 1575–1590 (2002). this method from endogenous substances in plasma or from common benzodiazepines, inhalating anesthetics, lo- cal anesthetics, antibiotics, tricyclic antidepressants, or skeletal muscle relaxants. MILNACIPRAN HYDROCHLORIDE CHEMISTRY Assay 2 Dufresne et al. [2] described an GC-MS method with direct solid-phase microextraction (SPME) for simul- Milnacipran hydrochloride is a serotonin and noradre- taneous determination of fentanyl and midazolam in hu- naline reuptake inhibitor. Its chemical name is (±)-cis-2- man plasma. A Hewlett-Packard (HP) 6890 gas chromato- (aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarbox- graph equipped with a split/splitless capillary inlet sys- amide hydrochloride. Its molecular formula is tem containing a quartz-deactivated liner (0.75 mm i.d.) C H N O·HCl, with a molecular weight of 282.8 was operated in the splitless mode. The stationary phase 15 22 2 and a CAS number of 175131-61-0. was an HP-5MS fused-silica capillary column (30 m × 0.25 mm, 0.50 ␮m film thickness). The carrier gas was helium, and the injector temperature was set at 270◦C. The column METHOD temperature was initially set at 180◦C for 5 min, increased to 280◦Cat20◦C/min, and held at 280◦C for 12 min. Assay 1 Duverneuil et al. [1] developed an HPLC An HP5973 mass spectrometer was operated in the elec- method for therapeutic drug monitoring of fluoxetine, tron impact mode. Analytes were detected in the selected- norfluoxetine, sertraline, paroxetine, citalopram, fluvox- ion monitoring (SIM) mode at ions m/z 310 and 137 for amine, moclobemide, mirtazapine, milnacipran, toloxa- midazolam, m/z 245 and 189 for fentanyl, and m/z 289 tone, venlafaxine, desmethylvenlafaxine, and viloxazine. and 140 for sufentanyl. A liquid chromatograph consisting of a Spectra Sys- A stock solution of midazolam at 50 ␮g/mL was pre- tem model P1000XR quaternary gradient pump, mode pared in water, and its working solutions were prepared by AS3000 autoinjector with a 100 ␮L loop, and model diluting this stock solution with water. Working solutions 6000LP photodiode-array detector was used. The station- of fentanyl were prepared in water. A working solution of ary phase was a ThermoHypersil Hypurity C18 column P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

326 MINOCYCLINE HYDROCHLORIDE

(250 × 4.6 mm, 5 ␮m particle size) protected by a Ther- molecular weight of 493.9 and a CAS number of 13614-98- moHypersil C18 precolumn (4 × 4.4 mm, 5 ␮m particle 7. Minocycline hydrochloride is a yellow crystalline pow- size). Solvent A was 20 mM monobasic sodium phosphate der. Minocycline hydrochloride is sparingly soluble in wa- buffer (pH 3.8), and solvent B was acetonitrile. The content ter and slightly soluble in alcohol. It is soluble in solutions of acetonitrile of the mobile phase was linearly increased of alkali hydroxides and carbonates. Minocycline has pKa from 25% to 40% in 10 min and then to 44% in another 8 values of 5 and 9.5 for two amine groups and 2.8 and 7.8 min. The equilibrium time before the next sample injection for two hydroxyl groups. was 3 min. The flow rate was 1.0 mL/min. UV detection was performed at 220 nm for viloxazine, milnacipran, venlafaxine, desmethylvenlafaxine, norfluoxetine, fluoxetine, and METHOD sertraline; at 240 nm for moclobemide, toloxatone, citalopram; and fluvoxamine; and at 290 nm for mirtazapine Assay1 Jain [1] et al. described the development and val- and paroxetine. The injection volume was 60 ␮L. The total idation of a high-performance thin-layer chromatographic runtime was 18 min. (HPTLC) method for determination of minocycline in hu- Stock solutions of fluoxetine, norfluoxetine, sertraline, man plasma. A Camag TLC Scanner III in absorbance paroxetine, citalopram, fluvoxamine, moclobemide, mir- mode, a Camag Linomat V sample applicator fitted with ␮ tazapine, milnacipran, toloxatone, venlafaxine, desmethyl- a 100- L syringe, and a Camag twin-trough glass cham- × venlafaxine, and viloxazine at 1.0 g/L each were prepared ber (20 10 cm) were used. The stationary phase was × ␮ in methanol. Working solutions of these drugs were pre- an aluminum plate (20 10 cm) coated with 200- m pared by diluting these stock solutions with methanol. layers of Merck silicagel 60F254. Plates were sprayed Standards were prepared by spiking blank human plasma with 10% (wt/vol) aqueous disodium ethylenediaminete- with working solutions. Protriptyline at 10 mg/L was used traacetic acid (EDTA) solution, adjusted to pH 9.0 with as an internal standard. All stock solutions were stored at 10% (mass/vol) aqueous sodium hydroxide solution, dried − ◦ horizontally at room temperature for 1 h, and heated at 20 C. An aliquot of 1 mL of plasma, control, or standard ◦ was mixed with 50 ␮L of the internal standard and 200 110 C in an oven for 1 h before use. The mobile phase ␮L of 2 M sodium hydroxide solution, extracted with 7 mL consisted of methanol, acetonitrile, isopropanol, and water of hexane–isoamyl alcohol (98 : 2, vol/vol), shaken for 15 (95 : 4: 0.5 : 0.5, vol/vol/vol/vol). Densitometric scanning min, and centrifuged at 3000 g for 5 min. The organic phase was performed at 345 nm. was separated, mixed with 200 ␮L of 0.01 N hydrochlo- A stock solution of minocycline was prepared in ric acid, shaken for 15 min, and centrifuged at 3000 g methanol. Working solutions were prepared by dilution for 5 min. The aqueous layer was collected and assayed. with methanol. Standards and controls were prepared by Under these conditions, retention times for moclobemide, spiking blank human plasma with working solutions and − ◦ viloxazine, mirtazapine, milnacipran, desmethylvenlafax- stored at 20 C. ine, venlafaxine, toloxatone, citalopram, paroxetine, pro- Standards and controls (1 mL) were mixed with 1 mL triptyline, fluvoxamine, norfluoxetine, fluoxetine, and ser- of methanol, vortexed for 1 min, and centrifuged at 2000 g for 5 min. The supernatant was collected, evaporated to traline were 5.0, 6.0, 6.5, 7.5, 7.7, 8.2, 9.3, 11.9, 13.9, 14.4, ◦ 15.1, 16.4, 17.3, and 17.7 min, respectively. dryness at 37 C under a stream of nitrogen, and reconsti- ␮ ␮ Calibration curves for milnacipran were constructed tuted in 100 L of methanol. An aliquot of 1 Lofthis over the range from 25 to 1000 ng/mL. Correlation coeffi- solution was applied to the plate to establish a final cali- cients were 0.9997. The accuracy was 99.7%. The average bration range of 100–1200 ng/zone. Plasma samples were recovery of the drug from plasma was better than 78.6%. applied onto the plates as bands 5 mm wide and 10 mm Intraassay and interassay coefficients of variation were apart using a sample applicator at 150 nL/s, developed linearly with mobile phase in a twin-trough glass chamber less than 3.5% and 14.8%, respectively. Limits of detection ◦ and quantification were 3 and 15 ng/mL, respectively. that was previously saturated with mobile phase at 25 C and relative humidity 60 ±5% for 30 min with a tight lid. The development distance was 8 mm. Plates were then REFERENCE dried in a stream of air and scanned at 345 nm. Under these conditions, the RF value of minocycline was 0.32. 1. Duverneuil C, de la Grandmaison GL, de Mazancourt P, A linear relationship was obtained by linear least- et al., A high-performance liquid chromatography method with squares regression of peak areas of minocycline against its photodiode-array UV detection for therapeutic drug monitor- concentrations in the range of 100–1200 ng/zone.The cor- ing of the nontricyclic antidepressant drugs, Ther Drug Monit relation coefficient was greater than 0.997. Mean recovery 25: 565–573 (2003). of minocycline from plasma was 95.8%. Intraday and interday coefficients of variation were less than 3.49% and 4.14%, respectively. Limits of detection and quantification MINOCYCLINE HYDROCHLORIDE were 5.1 and 15.4 ng/zone, respectively.

CHEMISTRY REFERENCE

Minocycline is a tetracycline antibacterial. Its chemical 1. Jain GK, Jain N, Iqbal Z, et al., Development and validation of name is 6-demethyl-6-deoxy-7-dimethylaminotetracycline an HPTLC method for determination of minocycline in human hydrochloride. Its molecular is C23H27N3O7·HCl, with a plasma, Acta Chromatogr 19: 197–204 (2007). P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

MIRTAZAPINE 327

MINOXIDIL crystalline powder. Mirtazapine is practically insoluble in water, soluble in solvent ether, and sparingly soluble in CHEMISTRY n-hexane. It is freely soluble in methanol and toluene.

Minoxidil is an antihypertensive. Its chemical name is 2,6- diamino-4-piperidinopyrimidine-1-oxide. Its molecular for- METHODS mula is C9H15N5O, with a molecular weight of 209.2 and a CAS number of 38304-91-5. Minoxidil is a white or off- Assay 1 Duverneuil et al. [1] developed an HPLC method white crystalline powder. Minoxidil is slightly soluble in for therapeutic drug monitoring of fluoxetine, norflu- water and soluble in alcohol and propylene glycol. It is oxetine, sertraline, paroxetine, citalopram, fluvoxamine, practically insoluble in acetone, chloroform, ethyl acetate, moclobemide, mirtazapine, milnacipram, toloxatone, ven- and petroleum spirit. The drug has a pKa of 4.6. lafaxine, desmethylvenlafaxine, and viloxazine. A liquid chromatograph consisting of a Spectra System model P1000XR quaternary gradient pump, mode AS3000 au- METHOD toinjector with a 100-␮L loop, and model 6000 LP photodiode-array detector was used. The stationary phase Assay 1 Zarghi et al. [1] reported the determination of mi- was a ThermoHypersil Hypurity C column (250 × 4.6 noxidil in human plasma using an ion-pair HPLC method. 18 mm, 5 ␮m particle size) protected by a ThermoHypersil A Knauer system consisting of a model Wellchrom K1001 C precolumn (4 × 4.4 mm, 5 ␮m particle size). Solvent A pump, Rheodyne model 7125 injector, model K2501 UV 18 was 20 mM monobasic sodium phosphate buffer (pH 3.8), detector, and Eurochrom model 2000 integrator was em- and solvent B was acetonitrile. The content of acetonitrile ployed. The stationary phase was a ␮Bondapak C ana- 18 of the mobile phase was linearly increased from 25% to lytical column (150 × 4.6 mm, 4 ␮m particle size). The col- 40% in 10 min and then to 44% in another 8 min. The umn temperature was set at 50◦C. The mobile phase con- equilibrium time before the next sample injection was 3 sisted of 0.01 M monobasic sodium phosphate buffer and min. The flow rate was 1.0 mL/min. UV detection was per- acetonitrile (60 : 40, vol/vol) containing 2.5 mM sodium formed at 220 nm for viloxazine, milnacipram, venlafax- dodecyl sulfate (SDS) adjusted to pH 3.5. The flow rate ine, desmethylvenlafaxine, norfluoxetine, fluoxetine, and was 1 mL/min. UV detection was performed at 281 nm. sertraline; at 240 nm for moclobemide, toloxatone, citalo- Stock solutions of minoxidil (1 mg/mL) and propyl- pram, and fluvoxamine; and at 290 nm for mirtazapine paraben as internal standard (1 ␮g/mL) were prepared in and paroxetine. The injection volume was 60 ␮L. The total methanol and stored at 4◦C. A plasma sample (500 ␮L) was runtime was 18 min. mixed with 50 ␮L of internal standard, 250 ␮Lofborate Stock solutions of fluoxetine, norfluoxetine, ser- buffer (pH 9.5), and 500 ␮L of acetonitrile; vortexed for 30 traline, paroxetine, citalopram, fluvoxamine, moclobe- s; and centrifuged at 6000 rpm for 15 min. The supernatant mide, mirtazapine, milnacipram, toloxatone, venlafaxine, was collected and injected. The injection volume was 50 ␮L. desmethylvenlafaxine, and viloxazine at 1.0 g/L each were Under these conditions, retention times of minoxidil and prepared in methanol. Working solutions of these drugs propylparaben were 3.6 and 4.8 min, respectively. were prepared by diluting these stock solutions with Calibration curves were constructed over the range of methanol. Standards were prepared by spiking blank 2–100 ng/mL. Correlation coefficients were greater than human plasma with working solutions. Protriptyline at 0.999. The average recovery of minoxidil from plasma was 10 mg/L was used as an internal standard. All stock solu- 96.1%. Intraday and interday coefficients of variation were tions were stored at −20◦C. An aliquot of 1 mL of plasma, 3.1% and 3.6%, respectively. The limit of detection was control, or standard was mixed with 50 ␮Loftheinter- 0.5 ng/mL. Over 300 plasma samples were analyzed using nal standard and 200 ␮L of 2 M sodium hydroxide solu- this method. tion, extracted with 7 mL of hexane–isoamyl alcohol (98 : 2, vol/vol), shaken for 15 min, and centrifuged at 3000 g REFERENCE for 5 min. The organic phase was separated, mixed with 200 ␮L of 0.01 N hydrochloric acid, shaken for 15 min, 1. Zarghi A, Shafaati A, Foroutan SM, et al., Rapid determination and centrifuged at 3000 g for 5 min. The aqueous layer of minoxidil in human plasma using ion-pair HPLC, JPharm was collected and assayed. Under these conditions, reten- Biomed Anal 36: 377–379 (2004). tion times for moclobemide, viloxazine, mirtazapine, milnacipram, desmethylvenlafaxine, venlafaxine, toloxatone, citalopram, paroxetine, protriptyline, fluvoxamine, norflu- MIRTAZAPINE oxetine, fluoxetine, and sertraline were 5.0, 6.0, 6.5, 7.5, 7.7, 8.2, 9.3, 11.9, 13.9, 14.4, 15.1, 16.4, 17.3, and 17.7 min, CHEMISTRY respectively. Calibration curves for mirtazapine were constructed Mirtazapine is a noradrenergic and specific serotonergic over the range from 25 to 1000 ng/mL. Correlation coeffi- antidepressant. Its chemical name is (RS)-1,2,3,4,10,14b- cients were 0.9997. The accuracy was 103.0%. The average hexahydro2-methylpyrazino-[2,1-a]pyrido[2,3-c][2]ben- recovery of the drug from plasma was better than 101.2%. zazepine. Its molecular formula is C17H19N3,witha Intraassay and interassay coefficients of variation were molecular weight of 265.4 and a CAS number of 61337- less than 2.6% and 4.7%, respectively. Limits of detection 67-5. Mirtazapine occurs as a white to creamy white, and quantification were 3 and 25 ng/mL, respectively. P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

328 MITOTANE

Assay 2 Frahnert et al. [2] reported the analysis of 2. Frahnert C. Rao ML, Grasmader K, Analysis of eighteen an- mirtazapine in human serum by HPLC for therapeutic tidepressants, four atypical antipsychotics and active metabo- drug monitoring. A liquid chromatograph consisting of a lites in serum by liquid chromatography: A simple tool for Bischoff 2200 pump, a Bischoff SDU2003 solvent degasser, therapeutic drug monitoring, J Chromatogr B 794: 35–47 a Waters WISP 717 autosampler, and a Shimadzu SPD- (2003). 10AVP UV detector was used. The stationary phase was a Macherey–Nagel Nucleosil 100-5-Protect 1 analytical column (250 × 4.6 mm, 5 ␮m particle size). The mobile phase MITOTANE consisted of 25 mM monobasic potassium phosphate buffer (pH 7.0) and acetonitrile (60 : 40) and was isocratically CHEMISTRY delivered at 1 mL/min. UV detection was performed at 230 nm. The injection volume was 100 ␮L. Mitotane is an antineoplastic. Its chemical name is 1,1- A stock solution of mirtazapine at 1.0 mg/mL was pre- dichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane. Its pared in methanol. Working solutions were prepared by molecular formula is C14H10Cl4, with a molecular weight diluting this stock solution with water. Standards were of 320.0 and a CAS number of 53-19-0. Mitotane occurs prepared by spiking blank human serum with working so- as a white crystalline powder with a slight aromatic odor. lutions. Melperone at 3000 ng/mL was used as an internal Mitotane is practically insoluble in water and soluble in al- standard. An aliquot of 1 mL of serum sample or stan- cohol, ether, petroleum spirit, fixed oils, and fats. Mitotane dard was centrifuged at 13,000 g and at 4◦C for 10 min. should be stored in airtight containers and protected from An aliquot of 0.9 mL of the supernatant was separated; light. mixed with 0.1 mL of internal standard and 2.0 mL of 0.1 M monobasic potassium phosphate buffer (pH 6.0); loaded onto a Varian 3M-Empore extraction disk cartridge (3 mL) METHOD that was preconditioned with 1 mL of methanol followed by 1 mL of water; pulled through the cartridge; washed se- Assay 1 Andersen et al. [1] reported an HPLC method quentially with 1 mL of water, 1 mL of 1 M acetic acid, 1 mL for the analysis of mitotane and its metabolite in plasma. of n-hexane, 2 mL of n-hexane–ethyl acetate (1 : 1), and A Shimadzu system included a model LC9A quaternary 1 mL of methanol; eluted with 1 mL of 2-propanol–25% gradient pump, model CTO6A column oven, model SIL9A ammonium solution–dichloromethane (20 : 2: 78); evap- autoinjector, model SPD6AV variable-wavelength detector, orated to dryness; reconstituted with 250 ␮Lofacetoni- model DGU3A online degasser, and model CR6A Chro- trile and water (3 : 7); and assayed. Under these condi- matopac integrator. The stationary phase was a Supelcosil tions, retention times for melperone and mirtazapine were LC18 column (150 × 4.6 mm, 3 ␮m particle size) coupled 8.8 and 16.6 min, respectively. with a 20-mm Supelguard. The column temperature was Calibration curves for mirtazapine were constructed maintained at 50◦C. The mobile phase was composed of 50 over the range from 5 to 300 ng/mL. Correlation coef- mM monobasic potassium phosphate buffer (adjusted to ficients were 0.9994. Recovery of the drug from serum pH 7.0 with potassium hydroxide) and methanol (20 : 80, ranged from 94.7% to 101.0%. Intraassay and interassay vol/vol). The flow rate was 1.25 mL/min. UV detection was coefficients of variation were less than 5.6% and 7.7%, carried out at 230 nm. respectively. There was no interference with the assay Stock solutions of mitotane and its metabolite were pre- from the following drugs and their metabolites (retention pared in ethanol. Standards were prepared by spiking times in minutes): sulpiride (4.1), O-desmethylvenlafaxine blank plasma with stock solutions. Plasma samples and (4.8), 9-OH-risperidone (6.6), m-chlorophenylpiperazine standards (200 ␮L each) were mixed with 300 ␮Loface- (8.0), normirtazapine (8.3), zolpidem (10.2), nordoxepin tone, vortexed, and centrifuged at 17,000 rpm for 5 min. (10.9), diazepam (11.0), benperidol (11.5), normaproti- The supernatant was collected and assayed. The injection line (11.5), dibenzepine (11.5), opipramol (11.6), nor- volume was 50 ␮L. Under these conditions, mitotane and fluoxetine (13.4), norclozapine (14.4), haloperidol (15.3), its metabolite eluted in 6.2 and 9.3 min, respectively. norclomipramine (19.2), trifluperidol (20.8), olanzapine Calibration curves for mitotane and its metabolite were (21.0), ziprasidone (26.4), promethazine (28.1), flupenazine linear in the range of 1–100 ␮M. The correlation coeffi- (31.0), nefazodone (32.5), chlorprothixene (36.4), thiori- cients were 0.9999 and 0.9998 for mitotane and its metabo- dazine (43.2), pimozide (44.1), carbamazepine, perazine, lite, respectively. Within-run and between-run coefficients zotepine, valproate, zopiclone, buspirone, lorazepam, and of variation were less than 4%. The limit of detection for biperidene. mitotane was 0.25 ␮M.

REFERENCES REFERENCE

1. Duverneuil C, de la Grandmaison GL, de Mazancourt P, 1. Andersen A, Warren DJ, Nome O, et al., A high-pressure liq- et al., A high-performance liquid chromatography method with uid chromatographic method for measuring mitotane [1, 1- photodiode-array UV detection for therapeutic drug monitor- (o,p-dichlorodiphenyl)-2,2-dichloroethane] and its metabolite ing of the nontricyclic antidepressant drugs, Ther Drug Monit 1,1-(o,p-dichlorodiphenyl)-2,2-dichloroethene in plasma, Ther 25: 565–573 (2003). Drug Monit 17: 526–531. (1995). P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

MOCLOBEMIDE 329

MOCLOBEMIDE Calibration curves for moclobemide were constructed over the range from 25 to 1000 ng/mL. Correlation coeffi- CHEMISTRY cients were 0.998. The accuracy was 99.2%. The average recovery of the drug from plasma was better than 59.1%. Moclobemide is a reversible inhibitor of monoamine Intraassay and interassay coefficients of variation were oxidase type A. Its chemical name is 4-chloro-N-(2- less than 2.0% and 5.9%, respectively. Limits of detection morpholinoethyl)benzamide. Its molecular formula is and quantification were 3 and 25 ng/mL, respectively. C13H17ClN2O2, with a molecular weight of 268.7 and a CAS number of 71320-77-9. Assay 2 Frahnert et al. [2] reported the analysis of moclobemide in human serum by HPLC for therapeutic METHODS drug monitoring. A liquid chromatograph consisting of a Bischoff 2200 pump, a Bischoff SDU2003 solvent de- Assay 1 Duverneuil et al. [1] developed an HPLC gasser, a Waters WISP 717 autosampler, and a Shimadzu method for therapeutic drug monitoring of fluoxetine, SPD10AVP UV detector was used. The stationary phase norfluoxetine, sertraline, paroxetine, citalopram, fluvox- was a Macherey–Nagel Nucleosil 100-5-Protect-1 analyti- amine, moclobemide, mirtazapine, milnacipram, toloxa- cal column (250 × 4.6 mm, 5 ␮m particle size). The mobile tone, venlafaxine, desmethyl venlafaxine, and viloxazine. phase consisted of 25 mM monobasic potassium phosphate A liquid chromatograph consisting of a Spectra System buffer (pH 7.0) and acetonitrile (60 : 40) and was isocrat- model P1000XR quaternary gradient pump, mode AS3000 ically delivered at 1 mL/min. UV detection was performed autoinjector with a 100-␮L loop, and model 6000LP at 230 nm. The injection volume was 100 ␮L. photodiode-array detector was used. The stationary phase A stock solution of moclobemide at 1.0 mg/mL was pre- was a ThermoHypersil Hypurity C18 column (250 × 4.6 pared in methanol. Working solutions were prepared by mm, 5 ␮m particle size) protected by a ThermoHypersil C18 diluting this stock solution with water. Standards were precolumn (4 × 4.4 mm, 5 ␮m particle size). Solvent A was prepared by spiking blank human serum with working so- 20 mM monobasic sodium phosphate buffer (pH 3.8), and lutions. Melperone at 3000 ng/mL was used as an internal solvent B was acetonitrile. The content of acetonitrile of the standard. An aliquot of 1 mL of serum sample or standard ◦ mobile phase was linearly increased from 25% to 40% in was centrifuged at 13,000 g and at 4 C for 10 min. An 10 min and then to 44% in another 8 min. The equilibrium aliquot of 0.9 mL of the supernatant was separated; mixed time before the next sample injection was 3 min. The flow with 0.1 mL of internal standard and 2.0 mL of 0.1 M rate was 1.0 mL/min. UV detection was performed at 220 monobasic potassium phosphate buffer (pH 6.0); loaded nm for viloxazine, milnacipram, venlafaxine, desmethyl- onto a Varian 3M-Empore extraction disk cartridge (3 mL) venlafaxine, norfluoxetine, fluoxetine, and sertraline; at that was preconditioned with 1 mL of methanol followed 240 nm for moclobemide, toloxatone, citalopram, and flu- by 1 mL of water; pulled through the cartridge; washed voxamine; and at 290 nm for mirtazapine and paroxetine. sequentially with 1 mL of water, 1 mL of 1 M acetic acid, The injection volume was 60 ␮L. The total runtime was 1mLofn-hexane, 2 mL of n-hexane–ethyl acetate (1 : 1), 18 min. and 1 mL of methanol; eluted with 1 mL of 2-propanol–25% Stock solutions of fluoxetine, norfluoxetine, ser- ammonium solution–dichloromethane (20 : 2: 78); evapo- traline, paroxetine, citalopram, fluvoxamine, moclobe- rated to dryness; reconstituted with 250 ␮L of acetonitrile mide, mirtazapine, milnacipram, toloxatone, venlafaxine, and water (3 : 7); and assayed. Under these conditions, desmethylvenlafaxine, and viloxazine at 1.0 g/L each were retention times for melperone and moclobemide were 8.8 prepared in methanol. Working solutions of these drugs and 5.6 min, respectively. were prepared by diluting these stock solutions with Calibration curves for moclobemide were constructed methanol. Standards were prepared by spiking blank hu- over the range from 50 to 1500 ng/mL. Correlation co- man plasma with working solutions. Protriptyline at 10 efficients were 0.9998. Recovery of the drug from serum mg/L was used as an internal standard. All stock solu- ranged from 98.6% to 99.0%. Intraassay and interassay tions were stored at −20◦C. An aliquot of 1 mL of plasma, coefficients of variation were less than 2.8% and 4.1%, control, or standard was mixed with 50 ␮Loftheinter- respectively. There was no interference with the assay nal standard and 200 ␮L of 2 M sodium hydroxide solu- from the following drugs and their metabolites (retention tion, extracted with 7 mL of hexane–isoamyl alcohol (98 : times in minutes): sulpiride (4.1), O-desmethylvenlafaxine 2, vol/vol), shaken for 15 min, and centrifuged at 3000 g (4.8), 9-OH-risperidone (6.6), m-chlorophenylpiperazine for 5 min. The organic phase was separated, mixed with (8.0), normirtazapine (8.3), zolpidem (10.2), nordoxepin 200 ␮L of 0.01 N hydrochloric acid, shaken for 15 min, (10.9), diazepam (11.0), benperidol (11.5), normaproti- and centrifuged at 3000 g for 5 min. The aqueous layer line (11.5), dibenzepine (11.5), opipramol (11.6), nor- was collected and assayed. Under these conditions, reten- fluoxetine (13.4), norclozapine (14.4), haloperidol (15.3), tion times for moclobemide, viloxazine, mirtazapine, mil- norclomipramine (19.2), trifluperidol (20.8), olanzapine nacipram, desmethylvenlafaxine, venlafaxine, toloxatone, (21.0), ziprasidone (26.4), promethazine (28.1), flupenazine citalopram, paroxetine, protriptyline, fluvoxamine, norflu- (31.0), nefazodone (32.5), chlorprothixene (36.4), thiori- oxetine, fluoxetine, and sertraline were 5.0, 6.0, 6.5, 7.5, dazine (43.2), pimozide (44.1), carbamazepine, perazine, 7.7, 8.2, 9.3, 11.9, 13.9, 14.4, 15.1, 16.4, 17.3, and 17.7 min, zotepine, valproate, zopiclone, buspirone, lorazepam, and respectively. biperidene. P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

330 MONTELUKAST SODIUM

REFERENCES between-day coefﬁcients of variation were 2.5% and 5.4% in urine and 2.4% and 3.5% in plasma, respectively. The 1. Duverneuil C, de la Grandmaison GL, de Mazancourt P, limit of detection was 0.1 ␮g/mL. et al., A high-performance liquid chromatography method with photodiode-array UV detection for therapeutic drug monitoring of the nontricyclic antidepressant drugs, Ther Drug Monit REFERENCE 25: 565–573 (2003). 2. Frahnert C. Rao ML, Grasmader K, Analysis of eighteen an- 1. Schwertner HA, Kong SB, Determination of modaﬁnil in tidepressants, four atypical antipsychotics and active metabo- plasma and urine by reversed phase high-performance lites in serum by liquid chromatography: A simple tool for ther- liquid-chromatography, J Pharm Biomed Anal 37: 475–479 apeutic drug monitoring, J Chromatogr B 794: 35–47 (2003). (2005).

MODAFINIL MONTELUKAST SODIUM CHEMISTRY CHEMISTRY Montelukast is a selective leukotriene receptor antag- Modaﬁnil is a central stimulant. Its chemical name is onist. Its chemical name is sodium 1-[({(R)-m-[(E)-2- 2-[(diphenylmethyl)sulﬁnyl]acetamide. Its molecular for- (7-chloro-2-quinolyl)vinyl]-␣-[o-(1-hydroxy-1-methylethyl) mula is C H NO S, with a molecular weight of 273.4 and 15 15 2 phenethyl]benzyl}thio)-methy]cyclopropaneacetate. Its a CAS number of 68693-11-8. molecular formula is C35H35ClNNaO3S, with a molecular weight of 608.2 and a CAS number of 151767-02-1. METHOD

Assay 1 Schwertner and Kong [1] described the determi- METHOD nation of modafinil in human plasma and urine by HPLC. A Waters liquid chromatograph consisting of a model Assay 1 Smith et al. [1] reported an HPLC method for 600E controller, model 717 autosampler, and model 996 the determination of montelukast in human plasma. A photodiode-array detector was used. The stationary phase Shimadzu liquid chromatograph consisting of a model was a Waters Symmetry C18 column (250 × 4.6 mm). The SCL10AVP controller, model SIL10ADVP autoinjector, mobile phase consisted of methanol, water, and acetic acid model LC10ADVP pump, model DGU14A solvent degasser, (500 : 500 : 1, vol/vol/vol) and was isocratically delivered a Sys-Tec column heater, and a Jasco FP-920 fluorescence at 1.0 mL/min. UV detection was performed at 233 nm. detector was used. The stationary phase was a Jones Chro- × The injection volume was 10–30 ␮L. matography Genesis Lighting C18 column (100 3.0 mm, A stock solution of modafinil at 1.0 mg/mL was prepared 3 ␮m particle size). The mobile phase consisted of ace- in methanol and stored at 4◦C. Standards were prepared by tonitrile, water, and glacial acetic acid (75 : 25 : 0.25, spiking drug-free human plasma or urine with this stock vol/vol/vol) and was isocratically delivered at 0.5 mL/min. solution. A stock solution of 3-(phenylthio)acetic acid at 1.0 The fluorescence detection was performed at an excitation mg/mL in methanol was used as an internal standard. An wavelength of 350 nm and an emission wavelength of 400 aliquot of 5 mL of urine sample or standard was extracted nm for cis-isomer and at an excitation wavelength of 350 with 10 mL of ethyl acetate, shaken for 30 min, and cen- nm and an emission wavelength of 440 nm for trans-isomer trifuged at 3000 rpm for 20 min. The extract was spiked of montelukast, respectively. with 50 ␮L of the internal standard, evaporated to dryness Stock solutions of montelukast at 200 ␮g/mL were at 60◦C under a stream of nitrogen, reconstituted with 700 prepared in methanol. Working solutions were prepared ␮L of mobile phase, vortexed for 10 s, centrifuged at 3000 by diluting stock solutions with acetonitrile and water rpm for 5 min, and assayed. For plasma, an aliquot of (75 : 25, vol/vol). Stock solutions and working solutions ◦ 2 mL of plasma sample or standard was spiked with 20 ␮L were stored at −70 C. Standards were prepared by spiking of the internal standard, vortexed for 10 s, extracted with blank human plasma with working solutions. An aliquot of 10 mL of a mixture of ethyl acetate and acetic acid (100 : 100 ␮L of plasma sample, control, or standard was mixed 1, vol/vol), shaken for 30 min, centrifuged at 3000 rpm for with 300 ␮L of acetonitrile, vortexed for 1 min, and cen- 20 min, evaporated to dryness at 60◦C under a stream of trifuged at 2500 rpm for 5 min. The supernatant was sepa- nitrogen, reconstituted with 700 ␮L of mobile phase, vor- rated and assayed. Under these conditions, retention times texed for 10 s, centrifuged again for 5 min, and assayed. for cis-andtrans-isomers of montelukast were 6.1 and Under these conditions, retention times for modafinil 8.1 min, respectively. and the internal standard were 11.5 and 15.2 min, Calibration curves for montelukast were constructed respectively. over the range from 5 to 2000 ng/mL. The accuracy ex- Calibration curves for modafinil were constructed over pressed as the relative percentage error was less than the range from 0.1 to 20.0 ␮g/mL. Correlation coefficients 9.1%. Recovery of the drug from plasma was greater than were greater than 0.999. Recovery of the drug was 80.0% 94.3%. Intraassay and interassay coefficients of variation in urine and 98.9% in plasma, respectively. Within-day and were less than 6.7% and 6.1%, respectively. P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

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REFERENCE fluorescence detection. A Gilson liquid chromatograph consisting of two model 302 pumps, a model 802 manometric 1. Smith GA, Rawls CM, Kunka RL, An automated method for the module, a model 811 dynamic mixer, a model 620 integra- determination of montelukast in human plasma using dual- tor, and a Rheodyne model 7125 injector was used. The sta- column HPLC analysis and peak height summation of the par- tionary phase was a Bischoff Spherisorb column (250 × 4.6 ent compound and its photodegradation product, Pharm Res mm, 5 ␮m particle size). The mobile phase consisted of hex- 21: 1539–1544 (2005). ane, isopropanol, and ammonia (95 : 4.5, 0.5, vol/vol/vol) and was isocratically delivered at 2 mL/min. Fluores- MORPHINE cence detection was performed at excitation wavelengths of 330–380 nm and emission wavelengths of 410–500 nm. The injection volume was 20 ␮L. CHEMISTRY Human hair samples of 4 cm in length and 100–200 mg in weight were washed with 10 mL of ethyl ether and Morphine is an opioid analgesic. Its chemical name is 12 mL of 0.01 M hydrochloric acid, incubated with 3 7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol. Its mL of either 0.1 M hydrochloric acid at 45◦Cfor12h, molecular formula is C H NO , with a molecular weight 17 19 3 0.6 M hydrochloric acid at 120◦C for 30 min, or 0.6 M of 285.3 and a CAS number of 57-27-2. Morphine sulfate sodium hydroxide at 120◦C for 30 min; neutralized with occurs as white, feathery, silky crystals, cubical masses of equimolar volumes of 3 M sodium hydroxide or hydrochlo- crystals, or a white crystalline powder. Morphine sulfate ric acid; poured into an Analytical Systems Toxi-Tubes A; has solubilities of approximately 62.5 mg/mL in water and ◦ rinsed with 2 mL of water; vortexed for 2 min; and cen- 1.75 mg/mL in alcohol at 25 C. Morphine has pK values a trifuged at 750 g for 10 min at room temperature. The of 8.31 for the amino group and 9.51 for the phenolic group ◦ organic phase was collected and the aqueous phase was at 25 C. Morphine sulfate should be stored in airtight con- reextracted with a mixture of methylene chloride, ethy- tainers and protected from light. lene chloride, and heptane (18 : 18 : 64, vol/vol/vol). The pooled organic phase was evaporated to dryness, dissolved METHODS in 50 ␮L of water, mixed with 50 ␮L of dansyl chloride at 1 g/L in acetone and 50 ␮Lof0.1Msodiumcarbonate Assay 1 Hong et al. [1] described the determination of solution, incubated at room temperature in the dark for morphine sulfate in human plasma using HPLC. A Shi- 90 min, extracted with 1 mL of toluene, and vortexed for madzu LC6A liquid chromatograph consisting of a model 2 min. The organic phase was evaporated to dryness un- LC6A isocratic pump, model CTO6A column oven, model der a stream of nitrogen, reconstituted with mobile phase, SPD6AV UV detector, model SCL6B controller, and model and assayed. Under these conditions, the retention time CR4A integrator was used. The stationary phase was a for morphine was about 13.5 min (estimated from the pub- Three Dimension Chromatography (China) YWG C col- 18 lished chromatogram). umn (250 × 4.6 mm, 10 ␮m particle size). The column tem- Calibration curves for morphine were constructed in perature was maintained at 45◦C. The mobile phase con- the range from 5 to 100 ng/mg hair. Correlation coeffi- sisted of methanol and 0.02 M phosphate buffer (pH 5.3) cients were 0.999. The mean recovery of morphine from (40 : 60) and was isocratically delivered at 1.0 mL/min. hair was 87.5%. Intraday and interday coefficients of vari- The injection volume was 20 ␮L. ation were 5.6% and 7.8%, respectively. The limit of detec- A stock solution of morphine sulfate at 0.2 mg/mL was tion was 60 pg. prepared in methanol. Working solutions were prepared by serial dilution of this stock solution with methanol. Stan- dards were prepared by spiking drug-free human plasma REFERENCES with working solutions. A stock solution of diazepam at 0.2 mg/mL was prepared in methanol and used as an in- 1. Hong Y, Dong W, Jin L, et al., HPLC determination of plasma ternal standard. An aliquot of 1.0 mL of plasma sample or morphine sulfate from oral ingestion of controlled release tablet standard was mixed with 25 ␮L of internal standard, vor- (Mescontine), Yaowu Fenxi Zazhi 18: 204–205 (1998). texed, loaded onto a C18 solid-phase extraction cartridge 2. Marigo M, Tagliaro F, Poiesi C, et al., Determination of mor- that was preconditioned, washed with 2 mL of water, dried, phine in the hair of heroin addicts by high performance liquid eluted with 2 × 2 mL of methanol, evaporated to dryness chromatography with fluorimetric detection, J Anal Toxicol 10: at 60◦C under a stream of nitrogen, reconstituted with 100 158–161 (1986). ␮L of mobile phase, and assayed. Under these conditions, retention times of diazepam and morphine sulfate were 8.56 and 12.03 min, respectively. Calibration curves for morphine sulfate were con- MOXIFLOXACIN structed over the range from 0.25 to 10.0 ␮g/mL. Corre- lation coefficients were 0.9994. Recovery of the drug from CHEMISTRY plasma was greater than 92.7%. Intraday and interday co- Moxifloxacin is a fluoroquinolone antibacterial. Its chemi- efficients of variation were 0.93% and 1.02%, respectively. cal name is 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy- Assay 2 Marigo et al. [2] reported the determination of 7-[(4␣S,7␣S)-octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl]- morphine in the hair of heroin addicts using HPLC with 4-oxo-3-quinolinecarboxylic acid hydrochloride. Its P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

332 MOXIFLOXACIN

◦ ◦ molecular formula is C21H24FN3O4·HCl, with a molecular 125 C, desolvation temperature 250 C, capillary voltage weight of 437.9 and a CAS number of 186826-86-8. 3.2 kV, cone voltage 20 V, and collision voltage 30 V. The selective ion monitoring (SIM) mode was used for quantification of moxifloxacin (m/z = 402) and the MRM mode, for METHODS its characterization. Stock solutions of antibiotics were prepared in acetoni- Assay 1 Schulte et al. [1] reported the determination of trile and 0.1% TFA (1 : 1, vol/vol). Working solutions were moxifloxacin in human plasma by HPLC with fluorescence prepared by diluting stock solutions with acetonitrile and detection. A Shimadzu system included a model LC6A distilled water (1 : 1, vol/vol). Standards were prepared pump and model RF353 fluorescence monitor. The sta- by spiking groundwater with working solutions. A surface tionary phase was a Macherey–Nagel Nucleosil 100-5 C 18 water sample or a standard (100 mL) was filtered through Nautilus column (125 × 4 mm, 5 ␮m particle size) pro- 0.45-␮m nylon membrane, adjusted to pH 3.0 with formic tected by a Macherey–Nagel Nucelosil 100-5 C Nautilus 18 acid, passed through an Oasis HLB cartridge that was CC8/4 precolumn. Solvent A was a mixture of acetonitrile preconditioned with 5 mL of methanol followed by 5 mL of and 0.01 M monobasic sodium phosphate (pH 2.7) (3 : 97, distilled water (acidified to pH 3.0 with formic acid), eluted vol/vol) and solvent B a mixture of acetonitrile and 0.01 M with 10 mL of methanol, evaporated to dryness in a water monobasic sodium phosphate (pH 2.7) (50 : 50, vol/vol). bath under a gentle stream of nitrogen, reconstituted with The mobile phase was delivered in a gradient mode con- 500 ␮L of water and acetonitrile (1 : 1), and assayed. The taining 9% acetonitrile initially and increasing to 90% ace- retention time for moxifloxacin was 17.54 min. tonitrile in 18 min. The flow rate was 1.5 mL/min. The A calibration curve for moxifloxacin was constructed fluorescence detector was operated at an excitation wave- in the concentration range of 10–500 ␮g/L. The correla- length of 295 nm and an emission wavelength of 440 nm. tion coefficient was greater than 0.9990. The coefficient of A plasma sample (400 ␮L) was mixed with 400 ␮Lof variation of the assay was less than 10%. The recovery of phosphate buffer containing 2 ␮g of levofloxacin as an in- moxifloxacin by SPE was 102%. Limits of detection and ternal standard, precipitated by 800 ␮L of acetonitrile, vor- quantification were 1.4 and 4.5 ␮g/L, respectively. texed for 30 s, and centrifuged at 3500 g for 10 min. The supernatant was collected and assayed. Under these conditions, retention times for levofloxacin and moxifloxacin Assay 3 Nemutlu et al. [3] reported the analysis of were 7.1 and 9.7 min, respectively. ciprofloxacin, enoxacin, levofloxacin, lomefloxacin, moxi- The calibration curve for moxifloxacin was linear in floxacin, oxolinic acid, and perfloxacin in plasma and am- the range of 0–3.5 ␮g/mL. The correlation coefficient was niotic fluid using HPLC. A Shimadzu system consisting of 0.9995. The mean extraction efficiency was 81%. Intraday a model 10ATVP solvent delivery system, model M10VP and interday coefficients of variation were less than 5.3% photodiode array detector, model SIL10ADVP autosam- and 4.1%, respectively. Limits of detection and quantifica- pler, and model CTO10ASVP oven was used. The station- × tion were 0.1 and 0.2 ␮g/mL, respectively. ary phase was a Zorbax Eclipse XDB C18 column (150 4.6 mm) protected by a Phenomenex C18 column (4 × 3.0 mm). ◦ Assay 2 Rao et al. [2] reported an HPLC-MS/MS The column temperature was maintained at 40 C. The mo- method for the simultaneous determination of norfloxacin, bile phase consisted of 15 mM citrate buffer adjusted to pH ciprofloxacin, levofloxacin, lomefloxacin, gatifloxacin, mox- 3.2 with 5 N sodium hydroxide, methanol, and acetonitrile ifloxacin, sparfloxacin, sulfamethoxazole, trimethoprim, and was delivered at 1.5 mL/min in a gradient mode: cefaclor, cefdinir, cefprozil, ceftiofur, cefadroxil, and cefuroxime axetil in surface waters. A Hewlett-Packard HP %50 mM Series 1100 liquid chromatograph was coupled with a Mi- Time (min) Citrate Buffer %Methanol %Acetonitrile cromass Quattro triple quadrupole mass spectrometer. The 0865 9 stationary phase was a GL Sciences reversed-phase In- 11 86 5 9 × ␮ ertsil ODS3V C18 column (250 4.6 mm, 5 m particle 12 75 5 20 size). The mobile phase consisted of 0.1% trifluoroacetic 18 75 5 20 acid (TFA) aqueous solution and acetonitrile and was de- 19 86 5 9 livered at 1.0 mL/min in a gradient mode: UV detection was performed at 280 nm. The injection vol- Time (min) %(0.1% TFA) %Acetonitrile ume was 20 ␮L. 08515 Stock solutions of ciprofloxacin and marbofloxacin (in- 15 65 35 ternal standard) at 500 ␮g/mL were prepared in 50 mM 20 20 80 acetic acid aqueous solution. Stock solutions of lev- 25 85 15 ofloxacin, enoxacin, lomefloxacin, oxolonic acid, and mox- 30 85 15 ifloxacin at 500 ␮g/mL were prepared in 0.05 M sodium hydroxide solution. A stock solution of perfloxacin at 500 The mass spectrometer was operated in the positive ␮g/mL was prepared in water/methanol (20 : 80, vol/vol). electrospray ionization mode. The operating conditions All stock solutions were stored at 4◦C in the dark. Work- were as follows: nebulizer gas 1.1 L/min, desolvation gas ing solutions containing all drugs were prepared by dilut- 11.3 L/min, nebulizer pressure 50 psi, source temperature ing stock solutions with water. Standards were prepared P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

MOXIFLOXACIN 333

by spiking blank human plasma and blank amniotic fluid Calibration curves for moxifloxacin were constructed with working solutions. over the range from 20 to 60 ␮g/mL. Correlation coeffi- An aliquot of 0.5 mL of a plasma sample, amniotic fluid, cients were 0.9991. Intraday and interday coefficients of or standard was mixed with internal standard, loaded variation were 3.2% and 3.7%, respectively. Limits of de- onto a Phenomenex Strata X (1 mL/30 mg) solid-phase tection and quantification were 2.7 and 8.8 ␮g/mL, respec- extraction cartridge that was preconditioned with 1 mL of tively. methanol followed by 1 mL of water, passed through the cartridge, washed with 2 × 1 mL of water, eluted with 2 mL Assay 5 Chan et al. [5] described the simultaneous deter- of 1% trifluoroacetic acid in a mixture of water and ace- mination of ofloxacin and moxifloxacin in human aqueous tonitrile (25 : 75, vol/vol), evaporated to dryness at 45◦C and vitreous humor by HPLC with fluorescence detection. under a stream of air, reconstituted with 200 ␮Lofmo- A Waters 600s liquid chromatograph, including a model bile phase, and assayed. Under these conditions, retention 626 pump, a model 717 autosampler, and a model 474 TM times of the internal standard, enofloxacin, levofloxacin, scanning fluorescence detector, was used. The stationary perfloxacin, ciprofloxacin, lomefloxacin, moxifloxacin, and phase was a Waters AccQ Tag amino acid analytical col- oxolonic acid were about 6.0, 6.9, 7.8, 8.7, 9.5, 11.3, 16.5, umn (150 × 3.9 mm, 4 ␮m particle size) protected by a Wa- and 18.0 min, respectively (estimated from the published ters Spherisorb ODS2 guard column (7.5 × 4.6 mm, 3 ␮m chromatogram). particle size). The column temperature was maintained at Calibration curves for moxifloxacin were constructed 30◦C. Mobile phase A consisted of acetonitrile, methanol, over the range from 0.05 to 30.00 ␮g/mL. Correlation coef- 0.05 M tetrabutylammonium chloride solution, and triflu- ficients were greater than 0.9991. The average recoveries oroacetic acid (37.5 : 12.5 : 949 : 1, pH 3.0) and mobile of moxifloxacin from plasma and amniotic acid were 96.5% phase B was a mixture of acetonitrile, methanol, 0.05 M and 94.7%. The accuracy in the relative percentage error tetrabutylammonium chloride solution, and trifluoroacetic was 1.2%. Intraday and interday coefficients of variation acid (75 : 25 : 899 : 1, pH 3.0). The mobile phase was de- were less than 4.4% and 7.9%, respectively. Limits of delivered at 1.0 mL/min in a gradient mode. Mobile phase A tection and quantification were 0.010 and 0.035 ␮g/mL, was delivered at 100% for the first 3 min, decreased lin- respectively. early to 0% from 3 to 12 min, held at 0% for 4 min, increased linearly from 0% to 100% in 1 min, and held at 100% for Assay 4 Gonzalez et al. [4] developed an HPLC-UV 5 min. The fluorescence detector was set at an excitation method for the simultaneous determination of cefepime, wavelength of 290 nm and an emission wavelength of 500 garenoxacin, moxifloxacin, and levofloxacin in human nm. The injection volume was 20 ␮L. urine. An Agilent system consisting of a model L7100 Stock solutions of ofloxacin and moxifloxacin at 10 mg/ pump, model G1315B diode-array detector, and a Rheo- mL were prepared in a mixture of methanol and water dyne model 7725i injector with a 20-␮L loop was used. (1 : 1). Standards were prepared by diluting the stock The stationary phase was a LiChrospher 100 RP18 column solution in mobile phase A containing sarafloxacin at 1 ␮g/ (250 × 4.6 mm, 5 ␮m particle size) protected by a precol- mL. Sarafloxacin was used as an internal standard. An umn (10 × 4.0 mm) of the same packing material. The aliquot of 10 ␮L of aqueous humor sample was mixed with mobile phase consisted of acetonitrile (A), 0.1 M phospho- 35 ␮L of the mobile phase A and 5 ␮L of sarafloxacin at ric acid/sodium hydroxide buffer (pH 3) (B), and 0.01 M 10 ␮g/mL, and assayed. For vitreous humor, an aliquot of n-octylamine solution (pH 3) (C) and was delivered in a 10 ␮L of sample was mixed with 35 ␮L of acetonitrile and 5 gradient mode as follows: ␮L of sarafloxacin at 10 ␮g/mL, centrifuged at 22,500 g for 15 min at 4◦C, evaporated to dryness under a stream of ni- Time (min) %A %B %C trogen, reconstituted with 50 ␮L of mobile phase A, and as- 0 8 46 46 sayed. Concentrations of ofloxacin and moxifloxacin in hu- 10 40 30 30 man plasma were determined as in vitreous humor. Under 12 40 30 30 these conditions, retention times of ofloxacin, sarafloxacin, 15 8 46 46 and moxifloxacin were 5.0, 12.0, and 16.7 min, respectively. Calibration curves for moxifloxacin were constructed The flow rate was 1.0 mL/min. UV detection was performed over the range from 10 ng/mL to 100 ␮g/mL. Correlation at 256 nm for cefepime, 292 nm for levofloxacin, 294 nm coefficients were greater than 0.996. The recovery of moxi- for moxifloxacin, and 282 nm for garenoxacin. The injection floxacin from humor was greater than 98.9%. The accuracy volume was 20 ␮L. was better than 94.1%. Intrabatch and interbatch coeffi- Stock solutions of cefepime, levofloxacin, moxifloxacin, cients of variation were less than 5.5% and 4.7% in aqueous and garenoxacin at 1 mg/mL were separately prepared humor and 6.7% and 5.1% in vitreous humor, respectively. in water, and working solutions were prepared by dilut- The limit of detection was 10 ng/mL. ing stock solutions with water. Standards were prepared by spiking blank human urine with working solutions. REFERENCES Plasma samples or standards were diluted with water, ␮ mixed, microfiltered through a 0.45- L filter, and assayed. 1. Schulte S, Ackermann T, Bertram N, et al., Determination of Under these conditions, retention times were 4.9, 7.5, 8.9, the newer quinolones levofloxacin and moxifloxacin in plasma and 10.7 min for cefempime, levofloxacin, moxifloxacin, by high-performance liquid chromatography with fluorescence and garenoxacin, respectively. detection, J Chromatogr Sci 44: 205–208 (2006). P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come

334 MYCOPHENOLATE MOFETIL

2. Rao RN, Venkateswarlu N, Narsimha R, Determination of an- pared by spiking drug-free human plasma with mycophe- tibiotics in aquatic environment by solid-phase extraction fol- nolic acid, its phenolic glucuronide metabolite, and acyl lowed by liquid chromatography–electrospray ionization mass glucuronide metabolite. An aliquot of 0.5 mL of plasma spectrometry, J Chromatogr A 1187: 151–164 (2008). sample or standard was acidified with 12.5 ␮L of 10% 3. Nemutlu E, Kir S, Ozy¨ unc¨ u¨ O,¨ et al., Simultaneous separation acetic acid, mixed with 1 mL of distilled water and 50 and determination of seven quinolones using HPLC: Analysis ␮L of the internal standard, loaded onto a Waters Sep-Pak of levofloxacin and moxifloxacin in plasma and amniotic fluid, plus C solid-phase extraction cartridge (360 mg/0.7 mL) Chromatographia 66: S15–S25 (2007). 18 that was preconditioned with 1.5 mL of acetonitrile, 3 mL 4. Gonzalez JAO, Mochon MC, Rosa FJB de la, Simultaneous of methanol, and 3 mL of water; washed with 500 ␮Lof determination of cefepime and the quinolones garenoxacin, water; eluted with 1.5 mL of 90% acetonitrile containing moxifloxacin, and levofloxacin in human urine by HPLC-UV, ␮ Microchim Acta 151: 39–45 (2005). 0.25% ammonia solution; mixed immediately with 25 Lof 10% acetic acid; evaporated to dryness; reconstituted with 5. Chan KP, Chu KO, Lai WWK, et al., Determination of ofloxacin ␮ and moxifloxacin and their penetration in human aqueous and 120 L of mobile phase; and assayed. Under these con- vitreous humor by using high-performance liquid chromatog- ditions, retention times of mycophenolic acid, its phenolic raphy fluorescence detection, Anal Biochem 353: 30–36 (2006). glucuronide metabolite, acyl glucuronide metabolite, and the internal standard were 11.9, 7.2, 12.9, and 14.4 min, respectively. Calibration curves for mycophenolic acid were con- ␮ MYCOPHENOLATE MOFETIL structed over the range from 0.05 to 50.0 g/mL. Correla- tion coefficients were greater than 0.9997. The extraction CHEMISTRY efficiency of mycophenolic acid from plasma was 89.1%. The accuracy ranged from 96.8% to 102.2%. Intraassay and Mycophenolate mofetil is an immunosuppressant. Its interassay coefficients of variation were less than 3.89% chemical name is 2-morpholinoethyl (E)-6-(4-hydroxy- and 6.45%, respectively. The limit of quantification was ␮ 6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-4- 0.05 g/mL. Calibration curves for mycophenolic acid phenolic glu- hexenoate. Its molecular formula is C23H31NO7,with a molecular weight of 433.5 and a CAS number of curonide metabolite were constructed over the range from ␮ 115007-34-6. Mycophenolate mofetil occurs as a white or 0.10 to 400 g/mL. Correlation coefficients were greater almost white, crystalline powder. Mycophenolate mofetil than 0.9996. The extraction efficiency of this metabolite is practically insoluble in water and sparingly soluble in from plasma was 97.5%. The accuracy ranged from 95.1% dehydrated alcohol. It is freely soluble in acetone. to 101.2%. Intraassay and interassay coefficients of variation were less than 5.34% and 2.92%, respectively. The limit of quantification was 0.10 ␮g/mL. METHOD Calibration curves for mycophenolic acid acyl glucuronide metabolite were constructed over the range from Assay 1 Mino et al. [1] described the simultaneous de- 0.08 to 8.00 ␮g/mL. Correlation coefficients were greater termination of mycophenolic acid and its glucuronides in than 0.9997. The extraction efficiency of this metabolite human plasma using HPLC. A Shimadzu system consist- from plasma was 101.9%. The accuracy ranged from 99.3% ing of a model LC10AT pump, model SIL10AXL autoin- to 100.2%. Intraassay and interassay coefficients of vari- jector, and model SPDM10AVP photodiode-array detector ation were less than 3.82% and 6.07%, respectively. The was used. The stationary phase was a TSKgel ODS-80T limit of quantification was 0.08 ␮g/mL. column (150 × 4.6 mm, 5 ␮m particle size). The column There was no interference with this assay from endoge- ◦ temperature was maintained at 40 C. The autosampler nous materials in plasma. was set at 4◦C. The mobile phase consisted of 30 mM tetra- n-butylammonium bromide solution containing 5 mM ammonium acetate (pH 9.0) and acetonitrile (67 : 33, vol/vol) REFERENCE and was isocratically delivered at 1 mL/min. UV detection ␮ was performed at 250 nm. The injection volume was 30 L. 1. Mino Y, Naito T, Matsushita T, et al., Simultaneous deter- A stock solution of carboxy butoxy ether derivative of mination of mycophenolic acid and its glucuronides in human mycophenolic acid at 100 ␮g/mL in 50% acetonitrile in wa- plasma using isocratic ion pair high-performance liquid chro- ter was used as an internal standard. Standards were pre- matography, J Pharm Biomed Anal 46: 603–608 (2008). 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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