High-throughput methods for determining pharmacological levels of plasma D-serine
Described herein are high-throughput methods of monitoring D-serine levels in plasma. The assay involves the use of strong cation solid phase extraction (SPE) to isolate D-serine from plasma, followed by quantitation of D-serine using the D-amino acid oxidase- (DAAO-) catalyzed reaction. Also described are methods of screening for compounds that act as DAAO inhibitors.
1. A method of quantifying D-serine in plasma comprising the steps of:
(a) contacting a quantity of plasma with a quantity of a first acidic liquid, wherein the plasma comprises D-serine, thereby forming an acidified sample;
(b) contacting the acidified sample with a quantity of a cation exchange resin, thereby separating the acidified sample into a first component and a second component, wherein the first component comprises D-serine and is retained on the resin;
(c) rinsing the resin containing the retained first component with a quantity of a second acidic liquid;
(d) contacting the resin with a quantity of a first basic liquid, thereby eluting from the resin D-serine and forming a D-serine eluent;
(e) contacting the D-serine eluent with flavin adenine dinucleotide and a quantity of D-amino acid oxidase in the presence of a precursor to a fluorescence signal and a quantity of horse radish peroxidase; and
(f) detecting the fluorescence signal.
2. The method of claim 1 , wherein the quantity of plasma is from about 20 μL to about 80 μL.
3. The method of claim 1 , wherein the pH of the first acidic liquid is from about 1.0 to about 2.0.
4. The method of claim 1 , wherein the pH of second acidic liquid is from about 1.0 to about 2.0.
5. The method of claim 1 , further comprising, after step (c) and prior to step (d), the step of:
(c′) rinsing the resin containing the first component with a second basic liquid, wherein the second basic liquid has a pH from about 7.5 to about 9.5.
6. The method of claim 1 , wherein the first basic liquid has a pH from about 12 to about 14.
7. The method of claim 1 , wherein step (e) is carried out in a buffered solution.
8. The method of claim 7 , wherein the quantity of D-amino acid oxidase in the buffered solution is from about 20 units/mg to about 50 units/mg.
9. The method of claim 1 , further comprising, after step (e) and prior to step (f), the step of
(e′) providing a light source, wherein the wavelength of the light source is about 530 nm.
10. The method of claim 1 , wherein the precursor is Amplex Red.