scholarly journals A New Approach for Second-tier Analysis of Methylmalonic Acid in Dried Blood Spots using Liquid Chromatography Tandem Mass Spectrometry

2021 ◽  
Author(s):  
Bijo Varughese ◽  
Dnyanoba Madrewar ◽  
Sunil Kumar Polipalli ◽  
Siddarth Ramji ◽  
Seema Kapoor
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Methylmalonic Acid ◽  
Dried Blood Spots ◽  
Tandem Mass ◽  
Blood Spots ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Second Tier

Introduction: Inactivity or diminished activity of an enzyme Methylmalonyl-CoA mutase (a Cobalamin dependent) enzyme causes inborn error of metabolism named Methylmalonic Acidemia/Aciduria (MMA). Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) based method for the diagnosis of MMA in Newborn Screening (NBS), is often challenging due to the nonspecificity of propionylcarnitine (C3), a primary marker in routine NBS. Aim: To develop a Flow Injection Analysis (FIA) method for the second-tier estimation of Methylmalonic acid in the Dried Blood Spots (DBS) of primary NBS. Materials and Methods: A retrospective NBS study was conducted for a period of two years i.e. (November 2015 to November 2017) at the Paediatrics Research and Genetic Lab of Maulana Azad Medical College associated Lok Nayak Hospital, a Tertiary Care Centre in New Delhi, India. DBS samples were collected by heel- prick method and a second tier detection, quantification of methylmalonic acid was performed by LC-MS/MS on all samples with abnormal C3 levels in primary NBS. Multiple Reaction Monitoring (MRM) mode at m/z 117→73 for MMA and m/z 120→75 for MMA(IS) and isotopic dilution approach was followed for quantification. Results: Intra-assay and inter-assay precision and accuracy was determined at two different levels of MMA (LQC≅2.0 μmol/L and HQC≅10.0 μmol/L), respectively. The Coefficient of Variation (%) for intraday precision ranged between 5.27% to 8.9%. Similarly, for interday it ranged from 4.99% to 9.93%. The average accuracy (%) also falls within (105.4% and 106.1%) for interday and (105.9% and 106.7%) for intraday assay. Stability for samples during storage at different temperature i.e., (fresh, 2-8°C and -20°C) showed long term stability at -20°C storage. The assay was linear over a calibration range of (0.5 to 20.00 μmol/L). Conclusion: The outcome of the present data offers the confidence and reliability in the possible utility of this method for the definitive diagnosis and follows-up of MMA patients.

scholarly journals ANALYSIS OF 6-MERCAPTOPURINE AND 6-METHYLMERCAPTOPURINE IN DRIED BLOOD SPOTS USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY AND ITS APPLICATION IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS

2017 ◽  
Vol 10 (9) ◽  
pp. 120 ◽  
Author(s):  
Supandi Supandi ◽  
Yahdiana Harahap ◽  
Harmita Harmita ◽  
Rizka Andalusia ◽  
Marlina Ika
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Formic Acid ◽  
Maintenance Phase ◽  
Dried Blood Spots ◽  
Tandem Mass ◽  
Blood Spots ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

  Objective: To analyze and validate 6-mercaptopurine (6-MP) and 6-methylmercaptopurine (6-MMP) in dried blood spots (DBS) using liquid chromatography-tandem mass spectrometry (LC/MS-MS).Methods: Bio-sampling dried blood spot with DBS-CAMAG® paper diameter of 8 mm and extracted with acetonitrile-methanol (1:3) containing internal standard 5-fluorouracil (5-FU). Separation was performed with C18 column Acquity® 1.7 μm (2.1 mm × 100 mm), with a mobile phase mixture of 0.1% formic acid in water 0.1% formic acid in acetonitrile with gradient elution and flow rate 0.2 ml/min. Mass detection was Waters Xevo TQD with positive electrospray ionization (ESI) for 6-MP, 6-MMP and negative ESI for 5-FU in multiple reaction monitoring modes. The ions of 6-MP was detected at m/z 153.09->119.09, 6-MMP at m/z 167.17->126.03, and 5-FU at m/z 129.15->42.05.Results: This method fulfill the requirements of selectivity, linearity, lower limit of quantification, accuracy, precision, carry-over, matrix effects, and stability which refers to the european medicines agency (EMEA) guidelines. The linearity of 0.99 was 26-1000 ng/mL for 6-MP and 6-MMP, respectively. The validated method was applied to two childhood ALL maintenance phase. Retrieved 6-MP levels of 10.37 pmol/8×108 erythrocytes, respectively. The levels of 6-MMP gained 16.59 pmol/8×108 erythrocytes, respectively.Conclusion: The developed LC/MS-MS method is valid to analysis 6-MP and 6-MMP in DBS simultaneous in vitro according to EMEA guidelines. The method was successfully applied to authentic capillary blood samples from two childhood patients with ALL in the maintenance phase.

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