P1: TIX/OSW P2: TIX c12 JWBS042-Xu October 6, 2010 21:53 Printer Name: Yet to Come 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%.