Synchronized determination of sacubitril and valsartan with some co-administered drugs in human plasma via UPLC-MS/MS method using solid-phase extraction.

An accurate and sensitive UPLC-MS/MS method was developed and validated for the simultaneous estimation of the newly developed combination of sacubitril and valsartan and the co-administered drugs nebivolol, chlorthalidone and esomeprazole in human plasma. Solid-phase extraction was conducted for the purification and extraction of the drugs from human plasma. Chromatographic separation was carried out on an Agilent SB-C18 (1.8 µm, 2.1 × 50 mm) column using losartan as internal standard. Isocratic elution was applied using acetonitrile-0.1% formic acid in water (85: 15, v/v) as mobile phase. Detection was carried out using a triple-quadrupole tandem mass spectrometer using multiple reaction monitoring, at positive mode at m/z 412.23 → 266.19 for sacubitril, m/z 436.29 → 235.19 for valsartan, m/z 405.8 → 150.98 for nebivolol, m/z 346.09 → 198 for esomeprazole and a selected combination of two fragments m/z 423.19 → 207.14 and 423.19 → 192.2 for losartan (internal standard), and in negative ionization mode at m/z 337.02 → 190.12 for chlorthalidone. The method was linear over the concentration ranges 30-2,000 ng/ml for sacubitril, 70-2,000 ng/ml for valsartan, esomeprazole and chlorthalidone and 70-5,000 pg/ml for nebivolol. The developed method is sensitive and selective and could be applied for dose adjustment, bioavailability and drug-drug interaction studies.

Synchronized determination of the novel heart failure combination therapy containing sacubitril calcium and valsartan by a validated spectrofluorimetric method.

A sensitive and accurate spectrofluorimetric method was proposed for the determination of Sacubitril calcium and Valsartan simultaneously in binary mixture. The method was established on measuring the native fluorescence of Sacubitril calcium and Valsartan upon excitation at 240 nm in acetonitrile. The emission of Sacubitril calcium was measured at 615 nm. For the determination Valsartan a first derivative ratio method was employed to eliminate any spectral interference. The ratio emission spectra were achieved by dividing the emission spectra of various concentrations of Valsartan by the emission spectrum of Sacubitril calcium (100 ng/ml) then the first derivative of the obtained ratio emission spectra was recorded using the proper smoothing factor. The amplitude at 354.9 nm on the first derivative ratio emission spectrum was used to calculate the concentrations of Valsartan in presence of Sacubitril calcium. The method was linear over the concentration range 100-1000 ng/ml for both Sacubitril calcium and Valsartan. The mean accuracy values were found to be 99.32 ± 0.62 and 99.30 ± 0.70 for Sacubitril calcium and Valsartan, respectively. Statistical comparison between results obtained by the proposed method and a reported method for this drugs showed no significant difference. This developed method was used for the quantitative determination of Sacubitril calcium and Valsartan in both pure and pharmaceutical dosage form.

Development and Bio-Analytical Validation of Chromatographic Determination Method of Rufinamide in Presence of its Metabolite in Human Plasma.

Rufinamide (RF), antiepileptic drug, is biotransformed to inactive metabolite. Frequent plasma monitoring is required for dose adjustment. This work is concerned with the development and validation of a sensitive and selective RP-HPLC method for the quantitative determination of RF in spiked human plasma in the presence of its main metabolite. Lacosamide was selected as internal standard. Preparation of plasma samples involved precipitation of plasma proteins using methanol. Isocratic elution mode was applied and the chromatographic separation was performed on Prontosil CN column (5 µm, 250 × 4.6 mm). Good resolution was achieved using acetonitrile: water (10:90, v/v, adjusted with 0.01 N aqueous solution of o-phosphoric acid to pH = 3) as a mobile phase at a flow rate of 1 mL/min, and UV detection was carried out at 210 nm. Linearity was observed over the concentration range of 0.5-50 µg/mL of RF in plasma. Bio-analytical validation of the developed method was carried out in accordance to the European Medicines Agency guidelines. The accuracy ranged from 95.97 to 114.13%, and the coefficient of variation of the assay intra-day and inter-day precision did not exceed 10%. The samples were stable under the employed experimental conditions. In conclusion, the findings of the present study revealed its usefulness for therapeutic drug monitoring, assessment of drug pharmacokinetics and application for bioequivalence study.

Determination of Rufinamide in the Presence of 1-[(2,6-Difluorophenyl)Methyl]-1H-1,2,3-Triazole-4 Carboxylic Acid Using RP-HPLC and Derivative Ratio Methods as Stability Indicating Assays to Be Applied on Dosage Form.

BACKGROUND: Rufinamide is a triazole derivative that is structurally dissimilar to other marketed antiepileptic drugs, has been assumed a marketing authorization, by the European Union and FDA, for use as a complementary therapy for seizures associated with Lennox-Gastaut syndrome. OBJECTIVE: This work is concerned with development of two methods for determination of rufinamide (RUF) in presence of 1-[(2,6-difluorophenyl)methyl]-1H-1,2,3-triazole-4 carboxylic acid as its alkaline degradation product in dosage form. METHODS: The first method was capable of determing RUF in the presence of its alkaline degradation product and in dosage form. Kromasil C8 column and mobile phase consisting of acetonitrile-water (50:50, v/v) were used and UV detection at 210 nm. In the second method, first derivative ratio spectrophotometry, RUF was determined by measuring peak amplitude at 269.5 nm over 5-30 µg/mL. RESULTS: The linearity range of RUF was 10-90 µg/mL for HPLC method covering its therapeutic range with r2 = 0.9999. Forced degradation under alkaline conditions was carried out, the degradation product was isolated and its structure was confirmed. Both methods were validated in accordance to ICH guidelines. Statistical analysis revealed no significant difference between obtained results and reported ones. CONCLUSION: The present study is useful for therapeutic drug monitoring and routine analysis of RUF in quality control laboratories. HIGHLIGHTS: Kinetics of the alkaline degradation of RUF was studied by following the concentration of the remaining drug until complete degradation was achieved.

A validated RP-HPLC method for the evaluation of the influence of grapefruit juice on liver S9 activation of sacubitril.

Grapefruit juice inhibits esterase enzyme. Therefore, a possible interaction with ester prodrugs should be taken into consideration. In this study, the influence of grapefruit juice on sacubitril (SAC) rat liver S9 activation by esterase enzyme was evaluated. An RP-HPLC method was developed and validated for estimation of SAC in rat liver S9 fraction using a C18 Cyano column as stationary phase and acetonitrile-sodium di-hydrogen phosphate buffer (0.02 m, pH 4 adjusted by o-phosphoric acid, 40:60, v/v), as mobile phase at a flow rate of 1 mL/min and UV detection at 254 nm. The method was successfully applied to an in vitro study in which SAC was incubated with rat liver S9 fraction prepared from rats that had previously ingested grapefruit juice for a week. The calculated SAC concentration after incubation was compared with that of SAC incubated with rat liver S9 fraction from the rat control group. The statistical significance between the results of test and control incubation sets was assessed. In conclusion, the current study demonstrated that grapefruit juice decreased SAC hydrolysis, hence delaying its activation to sacubitrilat (active form) in gut lumen. Based on this food-drug interaction, it may be required that grapefruit juice should be consumed with caution in patients receiving SAC.

Determination of four antiepileptic drugs in plasma using ultra-performance liquid chromatography with mass detection technique.

Status epilepticus (SE) is considered the second most frequent neurological emergency. Its therapeutic management is performed using sequential antiepileptic drug regimens. Diazepam (DIA), midazolam (MID), phenytoin (PHT) and phenobarbital (PB) are four drugs of different classes used sequentially in the management of SE. A sensitive, selective, accurate and precise method was developed and validated for simultaneous determination of the four antiepileptic drugs in human plasma. Their separation and quantification were achieved using ultra-performance liquid chromatography (UPLC) with mass detection using carbamazepine as internal standard (IS). For the first three drugs and the IS, UPLC-MS/MS with electrospray ionization working in multiple reaction monitoring mode was used at the following transitions: m/z 285 → 193 for DIA; m/z 326 → 291 for MID; m/z 253 → 182 for PHT; and m/z 237 → 194, 237 → 192 for IS. For the fourth drug (PB), a molecular ion peak of PB [M + H] + at m/z 233 was used for its quantitation. The method was linear over concentration ranges 5-500 ng/mL for DIA and MID and 0.25-20 µg/mL for PHT and PB. Bioanalytical validation of the developed method was carried out according to European Medicines Agency guidelines. The developed method can be applied for routine drug analysis, therapeutic drug monitoring and bioequivalence studies.