DoE-Based Analytical-FMCEA for Enhanced AQbD Approach to MEER-RP-HPLC Method for Synchronous Estimation of 15 Anti-Hypertensive Pharmaceutical Dosage Forms.

BACKGROUND: In the recent scenario of green chemistry, the usage of organic solvents should be minimized in the development of analytical methods for the safety of the environment and analysts. OBJECTIVE: An RP-HPLC method has been developed as an economical and eco-friendly alternative to published RP-HPLC methods for the analysis of fixed-dose combinations (FDCs) of anti-hypertensive drugs, for saving time, resources, costs, and organic solvent. METHODS: The method has been developed through the implementation of an enhanced analytical quality by design (AQbD) approach using a design of experiment (DoE)-based analytical failure mode critical effect analysis (AFMCEA). The AFMCEA was performed by identifying potential analytical failure modes and assessing their risk using risk priority number ranking and filtering. The DoE-based AFMCEA was implemented for response surface analysis and mitigation of high-risk analytical failure modes by Box-Behnken design (BBD). The method operable design ranges and control strategy were set for lifecycle management of the developed method. RESULTS: The RP-HPLC method was developed using a Shimpack octadecyl silane (ODS) C18 column and acetonitrile-water (pH 6.2; 42:58 %, v/v). The method was validated as per ICH Q2 (R1) guidelines. The method was applied for the synchronous assay of 15 FDCs of anti-hypertensive drugs. CONCLUSION: The developed method has fulfilled the requirements of numerous published RP-HPLC and HPTLC methods. Hence, this method is a multipurpose chromatography method for synchronous estimation of FDC products of anti-hypertensive drugs. This method can be used as a multipurpose (M), economical (E), eco-friendly (E), and rapid (R), MEER-RP-HPLC for quality control of multiple FDCs of anti-hypertensive drugs in the pharmaceutical industry. HIGHLIGHTS: Development of a MEER-RP-HPLC method for synchronous estimation of 15 pharmaceutical dosage forms of anti-hypertensive drugs. Implementation of an enhanced AQbD approach using a DoE-based AFMCEA to develop the method which was applied to the assay of 15 anti-hypertensive dosage forms.

RANBP2 and CLTC are involved in ALK rearrangements in inflammatory myofibroblastic tumors.

Inflammatory myofibroblastic tumors (IMTs) are rare soft tissue tumors occurring primarily in children and young adults. ALK gene rearrangements have been identified in this neoplasm, with fusion of the ALK gene at 2p23 to a number of different partner genes. Metaphase cytogenetic analyses of these tumors have been relatively few, however, and may help to identify additional variant partners. We report on an IMT from a 2-year-old boy with a karyotype of 45,XY,der(2)inv(2)(p23q12)del(2)(p11.1p11.2),-22. FISH showed ALK-RANBP2 fusion in this tumor. The breakpoint was cloned and the fusion was confirmed, making this the third reported case of IMT with ALK-RANBP2 fusion. In addition, we identified the ALK fusion partner in a previously reported IMT with t(2;17)(p23;q23) as CLTC, a gene reported to be involved in four other IMTs, and showed that the breakpoint involved a novel ALK-CLTC fusion. FISH evaluation of nine other IMTs identified CLTC as the fusion partner in one additional case, but RANBP2 was not involved in the remaining eight IMTs, suggesting that the variant partners involved in ALK rearrangements in IMTs are diverse.

Increased risk for developmental delay in Saethre-Chotzen syndrome is associated with TWIST deletions: an improved strategy for TWIST mutation screening.

The majority of patients with Saethre-Chotzen syndrome have mutations in the TWIST gene, which codes for a basic helix-loop-helix transcription factor. Of the genetic alterations identified in TWIST, nonsense mutations, frameshifts secondary to small deletions or insertions, and large deletions implicate haploinsufficiency as the pathogenic mechanism. We identified three novel intragenic mutations and six deletions in our patients by using a new strategy to screen for TWIST mutations. We used polymerase chain reaction (PCR) amplification with subsequent sequencing to identify point mutations and small insertions or deletions in the coding region, and real-time PCR-based gene dosage analysis to identify large deletions encompassing the gene, with confirmation by microsatellite and fluorescence in situ hybridization (FISH) analyses. The size of the deletions can also be analyzed by using the gene dosage assay with "PCR walking" across the critical region. In 55 patients with features of Saethre-Chotzen syndrome, 11% were detected to have deletions by real-time gene dosage analysis. Two patients had a translocation or inversion at least 260 kb 3' of the gene, suggesting they had position-effect mutations. Of the 37 patients with classic features of Saethre-Chotzen syndrome, the overall detection rate for TWIST mutations was 68%. The risk for developmental delay in patients with deletions involving the TWIST gene is approximately 90% or eight times more common than in patients with intragenic mutations.