LC-MS/MS progress in newborn screening.

Newborn screening programs detect treatable disorders in infants before they become symptomatic. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has greatly increased the screening possibilities by monitoring levels of amino acids and acylcarnitines. After the initial screening step, LC-MS/MS can also be used in screening positive samples as a second tier test to differentiate between true and false positive samples. As the list of disorders screened for by LC-tandem MS increases, questions arise about screening for untreatable disorders, such as some lysosomal storage diseases (LSDs). For LSDs screening methods are being developed and tested more quickly than treatments are becoming available. This goes against one of the main tenets of newborn screening which requires that a treatment be available. LC-MS/MS can detect several disorders with a single injection, which is important in high throughput laboratories. Measuring different amino acids and acylcarnitines can be used to detect up to 45 different inherited disorders depending on how diseases are counted. The LSD assays are designed in a similar way to detect multiple disorders with common sample preparation and a single injection. The clinical implications of applying this technology to NBS on a large scale in many jurisdictions across the world are discussed.

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-aminobenzoylglutamate (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 d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6N 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 microL) 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 microL injection). Limit of detection (LOD) was 1 nmol/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 micromol/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+/-2.3 umol/L (mean+/-S.D.). This group also had total serum catabolites of 11.9+/-7.6 nmol/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+/-23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n=18) were 33.9+/-22.7 and 11.4+/-8.7 nmol/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.

Free and bound enantiomers of methadone and its metabolite, EDDP in methadone maintenance treatment: relationship to dosage?

OBJECTIVES: To determine the effects of metabolism and protein binding on the relationship between administered dose, blood levels of R methadone and biological response by measuring the free and protein-bound forms of the R and S enantiomers of methadone and EDDP, its metabolite. DESIGN AND METHODS: To measure free and total drug, trough levels were collected from 45 methadone clients. To measure free methadone, samples were filtered using ultrafiltration with a MW weight cut-off of 10,000 and extracted using liquid-liquid extraction. The solvent was evaporated and samples reconstituted in mobile phase for analysis by LC/MS/MS. Total analyte was determined by extracting unfiltered samples. Enantiomeric separation of methadone and EDDP was by chiral chromatography. RESULTS: The presence of unmetabolized methadone suggested that none of the patients were very fast metabolizers. R and S forms were metabolized at the same rate at all administered doses. Free R methadone levels correlated both with methadone dose and with the total amount of R methadone. The free fraction of R methadone (%free R) was higher at lower doses than at high doses, varied from 5 to 25% and was inversely proportional to the total dose of administered drug in a relationship that was logarithmic and non-linear. CONCLUSIONS: By measuring the free, biologically active form of the drug, we were unable to account for the large variations in dose required between different patients to prevent the onset of withdrawal symptoms. The reason for the large range in dosage may be multifactorial.

Determination of free and protein-bound methadone and its major metabolite EDDP: enantiomeric separation and quantitation by LC/MS/MS.

OBJECTIVE: To measure free and protein-bound R- and S-enantiomers of methadone and its major metabolite, 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in serum. METHODS: To determine free fraction, samples were filtered using ultrafiltration membranes with a molecular weight cut-off of 10,000 Da and extracted using liquid-liquid extraction. The solvent extract was evaporated and reconstituted in mobile phase for analysis by LC/MS/MS. Total analyte was determined by extracting unfiltered samples. Enantiomeric separation was by chiral chromatography. RESULTS: LC conditions resulted in baseline separation of R- and S-EDDP, and 85% resolution of methadone enantiomers. Precision of spiked specimens for both R- and S-methadone and R- and S-EDDP was less than 10% at 100 nM, and did not exceed 20% at 10 nM. CONCLUSIONS: Using minimal sample clean-up and a total instrument run-time of 10 min, a rapid, sensitive and highly specific method was developed for quantitation of free and total R- and S-enantiomers of methadone and EDDP.