Fast, Simple Method for the Analysis of Benzodiazepines in Meconium and an Inter-laboratory Method Comparison
A novel method for the quantitation of 10 commonly prescribed benzodiazepines and/or their metabolites in meconium was developed using enzymatic hydrolysis, Dispersive Pipette XTRaction (DPX) + SALLE, and LC-MS/MS analysis.
DPX + SALLE combines Dispersive Pipette XTRaction and SALLE (Salting-out Assisted Liquid-Liquid Extraction) for a novel cleanup mechanism. XTR Tips contain Weak Anion Exchange (WAX) for cleanup and salt (S) necessary for SALLE. This methodology can remove matrix interferences in less than one minute. The method was evaluated for linearity, precision, extraction efficiency, and limits of detection and quantitation. To test the validity of our method, a blind study was done with a collaborative laboratory including 35 meconium patient samples tested for ten benzodiazepines and/or metabolites.
Monitoring benzodiazepines in meconium is important for identifying potential health risks and treatment options for newborns. Meconium analysis is complex as a result of its heterogeneous composition. Meconium is a formulation of epithelial cells, mucus, lanugo, bile acids and salts, sugars, lipids, pancreatic and intestinal secretions, and more. When using liquid chromatography coupled with mass spectrometry, this difficult matrix can cause ion suppression or ion enhancement of the analytes of interest. Therefore, it is imperative to perform sample preparation to minimize these matrix effects. Reducing matrix effects with sample preparation involves extracting target analytes from the endogenous biological matrix, a mission that is often times consuming and labor intensive. Benzodiazepines are extensively metabolized, producing many glucuronide conjugates. Glucuronides often have poor LC-MS/MS sensitivity so enzymatic hydrolysis is employed to cleave the glucuronide moiety, leaving free parent compounds for improved detection.
|1||Add 25 mg Meconium + 215 μL H20 in vial
Vortex until homogenous
|2||Add 130 μL of mix: buffer, enzyme, internal standard and vortex
Hydrolyze for 1 hr at 55 °C
|3||Add 600 μL ACN, vortex and centrifuge
Transfer supernatant to clean vial
|4||Aspirate and dispense 2x with WAX-S Tips|
|5||Transfer 500 μL of ACN upper layer
Reconstitute in 100 μL of 10% methanol
DPX + SALLE
Dispersive Pipette XTRaction incorporates a loosely contained sorbent in a pipette tip between a frit at the bottom and a barrier at the top of the tip.
Aspirate and dispense steps mix sample solution and loose sorbent material. Added salt (S) assists in phase separation during extraction