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A rapid, simple and specific method for estimation of Mefenamic acid in human plasma was validated using Indomethacin as internal standard. The analyte and internal standard were extracted from plasma using simple solid phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mM Ammonium Acetate in Water and acetonitrile (20:80, v/v) and detected by tandem mass spectrometry in negative ion mode. The ion transition recorded in multiple reaction monitoring mode were m/z 240.1 196.0 for Mefenamic acid and m/z 356.1312.0 for internal standard. Linearity in plasma was observed over the concentration range 35.000 - 7000.000 ng/mL for Mefenamic acid. The cv of the assay was 4.89 % to 5.98 % and accuracy was 99.36 to 102.20 % Intra and Interday respectively at LLOQ level. The validated method was applied to bioequivalence study of 250 mg Mefenamic acid in 28 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range. In BA/BE studies Haemolysis effect can be accountable for analysis of drug estimation in plasma. Validation of LC-MS/MS assays includes an assessment of Haemolysis effects though the experiment. Identification of this effect in presence of drug can be helpful tool for estimation of assay in plasma during the course of subject analysis. Validation of the method to evaluate Haemolysis effect can easily prove the impact on drug estimation. Validation of drug estimation can be challenged through the Haemolysis samples which are unknown though Incurred sample reanalysis to compare the method ruggedness with respect to Haemolysis sample which are extracted directly from subjects (volunteers). Methodology can be applied to reduce Haemolysis effect through proper extraction techniques like SPE. We establish method with proper extraction techniques to reduce the Haemolysis effect and then challenge our method in subject analysis specifically for Haemolysis samples along with normal plasma samples. Summarize the assay value for combination drugs for Haemolysis samples and through ISR cross check the techniques used for validation is properly applied. Mefenamic acid is a non-steroidal anti-inflammatory drug used to treat pain, including menstrual pain. It is typically prescribed for oral administration. Mefenamic acid decreases inflammation (swelling) and uterine contractions by a still unknown mechanism. However it is thought to be related to the inhibition of prostaglandin synthesis. There is also evidence that supports the use of Mefenamic acid for perimenstrual migraine headache prophylaxis, with treatment starting 2 days prior to the onset of flow or 1 day prior to the expected onset of the headache and continuing for the duration of menstruation. Since hepatic metabolism plays a significant role in Mefenamic acid elimination, patients with known liver deficiency may be prescribed lower doses. Kidney deficiency may also cause accumulation of the drug and its metabolites in the excretory system. Therefore patients suffering from renal conditions should not be prescribed Mefenamic acid. It"s rapidly absorbed. T max is 2 to 4 h. C max is 20 mcg/mL (single doses). Steady state is reached in 2 days and Apparent Vd is 1.06 L/kg. Protein binding is more than 90%. Excreted in breast milk. Metabolized by CYP-450 enzyme CYP2C9 to 3-hydroxymethyl Mefenamic acid (metabolite I). Further oxidation to a 3-carboxyMefenamic acid (metabolite II) may occur. The metabolites may undergo glucuronidation, and Mefenamic acid is also glucuronidated directly. Approximately 52% of a dose is excreted into the urine primarily as glucuronides of Mefenamic acid (6%), 3-hydroxyMefenamic acid (25%), and 3- carboxyMefenamic acid (21%). The fecal route of elimination accounts for up to 20% of the dose, mainly in the form of unconjugated 3-carboxyMefenamic acid. The half-life is approximately 2 h. MEFENAMIC ACID is a member of the fenamate group of nonsteroidal anti-inflammatory drugs (NSAIDs). Each blue-banded, Mefenamic acid is a white to greyish-white, odorless, microcrystalline powder with a melting point of 230°-231°C and water solubility of 0.004% at pH 7.1. The chemical name is N-2,3- xylylanthranilic acid. The molecular weight is 241.29. Its molecular formula is C15H15N02 and the structural formula of Mefenamic acid is

Haemolysis effect of Mefenamic Acid 250 mg Capsule in Bio analysis by liquid chromatography - tandem mass spectrometry