Determination of molsidomine and its active metabolite in human plasma using liquid chromatography with tandem mass spectrometric detection.

Pharmacokinetic studies of molsidomine require a sensitive analytical method to allow the determination of concentrations of this compound and its active metabolite 3-morpholinosydnonimine (Sin-1) in the ng/ml range in plasma. The method developed is based on on-line LC-MS-MS using pneumatically assisted electrospray ionisation as an interface, preceded by off-line solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (automated sample preparation with extraction cartridges; ASPEC system). The DEC, filled with phenyl-modified silica, was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol containing formic acid (0.2%) and water. The liquid chromatographic separation of molsidomine and Sin-1 was achieved on an RP-8 stationary phase (5 microns). The mobile phase was a mixture of methanol-water-formic acid (65:35:0.1, v/v/v). The HPLC system was then coupled to a MS-MS system with an atmospheric pressure ionisation interface in the positive ion mode. The chromatographed analytes were detected in the multiple reaction monitoring mode. The MS-MS ion transitions monitored were (m/z) 243-->86 for molsidomine and 171-->86 for Sin-1. The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 74 +/- 3 and 55 +/- 5% for molsidomine and Sin-1, respectively. The method was found to be linear in the 0.5-50 ng/ml concentration range for the two analytes (r2 = 0.999 for both molsidomine and Sin-1). The mean RSD values for repeatability and intermediate precision were 3.4 and 4.8% for moldsidomine and 3.1-7.7% for the metabolite. The method developed was successfully used to investigate the bioequivalence of oral doses of molsidomine between a generic tablet and a reference product.

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry.

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.

Simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography-tandem mass spectrometry.

Quantitative analysis of therapeutic compounds and their metabolites in biological matrix (such as plasma, serum or urine) nowadays requires sensitive and selective methods to allow the determination of concentrations in the ng/ml range. A new on-line LC-MS-MS method using atmospheric pressure chemical ionisation (APCI) as interface for the simultaneous determination of nifedipine (NIF) and its metabolite in human plasma, dehydronifedipine (DNIF) has been developed. The compounds were extracted from plasma using solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with phenyl modified silica was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol and water. The liquid chromatographic (LC) separation of NIF and DNIF was achieved on a RP-18 stationary phase (4 microm). The mobile phase consisted of methanol-50 mM ammonium acetate solution (50:50, v/v). The LC was then coupled to tandem mass spectrometry with an APCI interface in the positive ion mode. The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 95+/-2% and 95+/-4% for NIF and DNIF, respectively. The method was found to be linear in the 0.5-100 ng/ml concentration range for the two analytes (r2 = 0.999 for both NIF and DNIF). The mean R.S.D. values for repeatability and intermediate precision were 2.9 and 3.0% for NIF and 2.2-4.7% for the metabolite. The method developed was successfully used to investigate the plasma concentration of NIF and DNIF in the pharmacokinetic studies.