Hengchang Song

Simultaneous determination of losartan and EXP3174 in human plasma and urine utilizing liquid chromatography/tandem mass spectrometry

Losartan is an orally active angiotensin II receptor antagonist indicated for the treatment of hypertension. EXP3174 is an active metabolite, which contributes to the overall activity of losartan. Analytical methods for the simultaneous determination of losartan and its active metabolite EXP3174 in human plasma and urine with limited plasma sample size have been developed and validated to support a pediatric clinical program. In both methods, analytes are extracted from the matrixes by liquid-liquid extraction and separated using reverse phase high-performance liquid chromatography (HPLC). A tandem mass spectrometer (MS/MS) with a Turbo ionspray (TIS) interface in multiple-reaction-monitoring (MRM) mode is used for detection of the analytes in both methods. The plasma method has a lower limit of quantitation (LOQ) of 1 ng/ml with a linearity range of 1-500 ng/ml for losartan and EXP3174 using 100 microl of plasma. For the urine method, the LOQ for both losartan and EXP3174 is 2 ng/ml using 0.5 ml of urine, and the linearity range for both analytes is 2-1000 ng/ml. Validation procedures have proven that both methods are robust, accurate, and reproducible. Both methods have been used to assay clinical samples and provided satisfactory results.

Simultaneous determination of a novel thrombin inhibitor and its two metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

I, 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylene-c arboxamidomethylpyridinyl)-pyrazinone dihydrochloride monohydrate, is a potent, orally active thrombin inhibitor. The compound also has two metabolites which have been shown to have thrombin inhibitory activity: benzylic alcohol M3 metabolite (II) and hydroxymethylpyrazinone M7 metabolite (III). A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of I and its two metabolites in human plasma has successfully been developed. The method consists of treating 0.5 ml plasma with 2 N NaOH and extracting I, II, III and internal standard (IV) with ethyl acetate:methyl-t-butyl ether (1:3, v/v). The analytes were then back-extracted into 2% formic acid. The analytes were chromatographed by high performance liquid chromatography (HPLC) and detected by MS/MS. Positive ionization was used on a heated nebulizer probe monitoring precursor --> product ion combinations in multiple reaction monitoring mode. The linear range is 0.5-1000 nM for I and III and 0.75-1000 nM for II. Recoveries were 88, 74, 78 and 102.1% for I, II and III and the internal standard, respectively in human plasma. Intraday variation using this method was < or = 7.9% for I, < or = 9.0%, for II and < or = 9.5% for III across the standard curve range. This method exhibits good linearity and reproducibility for the three analytes.

Absorption, metabolism, and excretion of [14C]mk-0767 (2-methoxy-5-(2,4-dioxo-5-thiazolidinyl)-N[[4-(trifluoromethyl)phenyl] methyl]benzamide) in humans

MK-0767 (KRP-297; 2-methoxy-5-(2,4-dioxo-5-thiazolidinyl)-N-[[4-(trifluoromethyl)phenyl] methyl]benzamide) is a thiazolidinedione (TZD)-containing dual agonist of the peroxisome proliferator-activated receptors α and γ that has been studied as a potential treatment for patients with type 2 diabetes. The metabolism and excretion of [14C]MK-0767 were evaluated in six human volunteers after a 5-mg (200 μCi) oral dose. Excretion of 14C radioactivity was found to be nearly equal into the urine (∼50%) and feces (∼40%). Elimination of [14C]MK-0767 was primarily by metabolism, with minimal excretion of parent compound into the urine (<0.5% of dose) and feces (∼14% of the dose). [14C]MK-0767 was the major circulating compound-related entity (>96% of radioactivity) through 48 h postdose. It was also found that ∼91% of the total radioactivity area under the curve was due to intact MK-0767. Several minor metabolites were detected in plasma (<1% of radioactivity, each), formed by cleavage of the TZD ring and subsequent S-methylation and oxidation. All the metabolites excreted into urine were formed by TZD cleavage, whereas the major metabolite in feces was the O-demethylated derivative of MK-0767.

Determination of a novel thrombin inhibitor in human plasma and urine utilizing liquid chromatography with tandem mass spectrometric and ultraviolet detection

An LC-MS-MS method and an HPLC-UV method have been developed for the assay of a novel thrombin inhibitor in human fluids. The LC-MS-MS method is developed for plasma, which usually requires maximum sensitivity. The HPLC-UV method is for urine. In both methods, analytes are extracted using liquid-liquid extraction, and analyzed by reversed-phase high-performance liquid chromatography. A tandem mass spectrometer in the multiple reaction monitoring (MRM) mode is used for detection of the analytes in the plasma method. UV is the detector for the urine method. The plasma method has a lower limit of quantitation (LOQ) of 0.1 ng/ml with a linearity range of 0.1-100 ng/ml. The urine method has an LOQ of 8.12 ng/ml (20 nM) and the linear dynamic range is 8.12-8120 ng/ml (20-20000 nM). Both methods are fast, specific and sensitive. Various validation procedures have proven that both methods are rugged, robust and reproducible. The research also suggested that, while LC-MS-MS provides superior sensitivity and selectivity for the determination of drugs and their metabolites at very low concentrations, HPLC with a conventional detector such as UV is still useful in the analysis when the sensitivity requirement is not crucial.

Simultaneous determination of a novel M3 muscarinic receptor antagonist and its active 5-OH metabolite in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive, specific, and robust liquid chromatography (LC)/mass spectrometry (MS)/MS method has been developed and validated for a novel M(3) muscarinic receptor antagonist (I) and its active 5-OH metabolite (II) in human plasma. The assay involves a two-step liquid-liquid extraction of the compounds from human plasma, high performance liquid chromatography (HPLC) separation, and MS/MS for the detection of the analytes. The method provides a linear response from a quantitation limit of 0.05-20 ng/ml for I and 0.1-20 ng/ml for II using 1 ml of plasma. The mean absolute recovery was 85.4% for I and 80.8% for II, respectively. The intra-assay accuracy of I and II averaged from 95.0 to 105.3% with coefficient of variation (CV) values <or=6.5% over the standard curve range. The stability study showed that I and II are stable in the plasma matrix over a period of 11 months at -70 degrees C. The accuracy, ruggedness, and reproducibility of this method were demonstrated by analyzing over 5000 plasma samples in clinical pharmacokinetics studies over a 6-month period.