Methods for assaying alpha-L-iduronidase enzymatic activity

A method for assaying α-L-iduronidase enzymatic activity, comprising:

• (a) contacting an α-L-iduronidase substrate with α-L-iduronidase for a pre-determined time to provide a solution comprising an α-L-iduronidase product;
• (b) contacting the α-L-iduronidase with an α-L-iduronidase internal standard before, simultaneously with, or after contacting the α-L-iduronidase substrate with α-L-iduronidase to provide a solution comprising the α-L-iduronidase product and α-L-iduronidase internal standard, wherein the internal standard has the formula
• wherein R is independently at each occurrence H or D and n is an integer from 1 to 20;
• (c) extracting the solution comprising the α-L-iduronidase product and α-L-iduronidase internal standard with an organic solvent to provide an organic phase that includes the α-L-iduronidase product and α-L-iduronidase internal standard;
• and
• (d) determining the quantity of the α-L-iduronidase product.

A method for screening newborns for Mucopolysaccharidosis Type-I, comprising:

• (a) contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution comprising α-L-iduronidase;
• (b) contacting an α-L-iduronidase substrate with the solution comprising α-L-iduronidase for a pre-determined time to provide a solution comprising an α-L-iduronidase product;
• (c) contacting the α-L-iduronidase with an α-L-iduronidase internal standard before, simultaneously with, or after contacting the α-L-iduronidase substrate with α-L-iduronidase to provide a solution comprising the α-L-iduronidase product and α-L-iduronidase internal standard, wherein the internal standard has the formula
• wherein R is independently at each occurrence H or D and n is an integer from 1 to 20;
• (d) extracting the solution comprising the α-L-iduronidase product and α-L-iduronidase internal standard with an organic solvent to provide an organic phase that includes the α-L-iduronidase product and α-L-iduronidase internal standard;
• (e) determining the quantity of the α-L-iduronidase product by tandem mass spectrometric analysis, comprising
• (i) generating, isolating, and subjecting the parent ions of the product and internal standard to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and
• (ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of α-L-iduronidase product;
• and
• (f) using the amount of α-L-iduronidase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis Type-I.