Simple method for the quantitative analysis of endogenous folate catabolites p-aminobenzoylglutamate (pABG) and its acetamido (apABG) derivative in human serum and urine by liquid chromatography-tandem mass spectrometry

Objective: To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylalutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Design and methods: Urine, serum and aqueous standards were thawed. Two microliters of d(3)-glutamic acid (d(3)-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6 N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 x g for 10 min and the supernatant (10 mu L) injected into a Biorad CAT/MET analytical column fitted to the LC-MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines. Results: pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 mu L injection). Limit of detection (LOD) was 1 mu mol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 Lmol/L for urines. Imprecision for spiked serum samples (n = 10) was between 2.5 and 20% for apABG and 4.5 and 21 % for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n = 10) was between 2.9 and 4.0% for apABG and 6.0-12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n=5) are 2.9:1-2.3umol/L (mean +/- E S.D.). This group also had total serum catabolites of 11.9 +/- 7.6nmol/L and serum folate of 35.3 +/- 5.8 nmol/L. Serum from patients being tested for PTH (n = 11) had serum folate levels of 27.0 +/- 10.4 nmol/L with total serum catabolites of 20.4 +/- 23.8nmol/L. Levels of serum folate and total catabolites in pregnant women (n = 18) were 33.9 +/- 22.7 and 11.4 +/- 8.7nmol/L. respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n = 19) was 581.8 +/- 368.4 nmol/L. Conclusion: A simple, reliable and highly specific method by LC-MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients. (c) 2006 Elsevier B.V. All rights reserved.