Internal standard tracked dilution to overcome challenges in dried blood spots and robotic sample preparation for liquid chromatography/tandem mass spectrometry assays

2011 ◽  
Vol 25 (9) ◽  
pp. 1250-1256 ◽  
Author(s):  
Guowen Liu ◽  
Heidi M. Snapp ◽  
Qin C. Ji
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Sample Preparation ◽  
Tandem Mass Spectrometry ◽  
Internal Standard ◽  
Dried Blood Spots ◽  
Tandem Mass ◽  
Blood Spots ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

scholarly journals DEVELOPMENT AND VALIDATION METHOD OF CYCLOPHOSPHAMIDE AND 4-HYDROXYCYCLOPHOSPHAMIDE WITH 4-HYDROXYCYCLOPHOSPHAMIDE-D4 AS INTERNAL STANDARD IN DRIED BLOOD SPOTS USING UPLC-MS/MS

2021 ◽  
pp. 148-152
Author(s):  
YESI IHDINA FITYATAL HASANAH ◽  
YAHDIANA HARAHAP ◽  
HERMAN SURYADI
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Analytical Method ◽  
Internal Standard ◽  
Dried Blood Spots ◽  
Tandem Mass ◽  
Blood Spots ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Objective: Cyclophosphamide (CP) is anticancer of the alkylating agent (nitrogen mustard) and a prodrug which will be metabolized into an active metabolite form, 4-hydroxycyclophosphamide (4-OHCP). Therefore, the effectiveness of therapy with CP is determined by its metabolites concentration. The purpose of this study was to obtain a validated analytical method of CP and 4-OHCP simultaneously and sensitively in dried blood spots with SIL (Stable Isotope Labeled) 4-OHCP-d4 as the internal standard using liquid chromatography-tandem mass spectrometry, so optimization and full validation are conducted in this research. Methods: A simpler analytical method was developed and validated to quantify CP and 4-OHCP in DBS samples using an Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). A linear regression was used as the statistical analysis method. Sample preparation was performed by protein precipitation using methanol. The separation was performed on UPLC H-Class BEH C18 column using formic acid 0.01%-acetonitrile as the mobile phase in gradient mode at 0.2 ml/minute. The mass detection was performed on Waters Xevo TQD using ESI+for CP, 4-OHCP-SCZ, and IS 4-OHCP-d4-SCZ with m/z value: 261.03>140.16; 334.10>221.04; and 338.10>225.06. Results: This method was linear within the range of 10–40,000 ng/ml for CP and 5–4,000 ng/ml for 4-OHCP. Lower Limit of Quantification (LLOQ) concentration of CP was 10 ng/ml and 4-OHCP was 5 ng/ml. Conclusion: This method has successfully fulfilled the validation requirement referring to the 2011 EMA and 2018 FDA guidelines.

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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