Rapid determination of C4-acylcarnitine and C5-acylcarnitine isomers in plasma and dried blood spots by UPLC-MS/MS as a second tier test following flow-injection MS/MS acylcarnitine profile analysis.

BACKGROUND: Flow-injection MS/MS methods for elevated acylcarnitines are routinely performed in most newborn screening and biochemical genetics laboratories; however this technique cannot distinguish between isobaric compounds; therefore, chromatographic separation is required to quantitate isomers for differential diagnosis of some inborn errors of metabolism. METHODS: A UPLC-MS/MS method has been developed for the simultaneous quantitation of isobutyrylcarnitine and butyrylcarnitine, and a second UPLC-MS/MS method for the quantitation of isovalerylcarnitine, (S) and (R) 2-methylbutyrylcarnitine, pivaloylcarnitine and valerylcarnitine. Plasma and dried blood spots samples are extracted with methanol and derivatized with butanolic HCl. Deuterium labeled internal standards are used for quantitation. Separation is obtained using a methanol/water gradient with a C18 BEH, 1x100mm, 1.7microm UPLC column, at 60 degrees C; run time is less than 10min. The isomers are detected with a Quattro Premier triple quadrupole, with electrospray ionization in positive ion mode. RESULTS: Intra-day precision in plasma and dried blood spots ranged from 1.4% to 14% and accuracy from 88% to 114% respectively for butyrylcarnitine and isobutyrylcarnitine. Precision for the isomers of C5-acylcarnitine ranged from 1.3% to 15% and accuracy 87% to 119%, respectively in plasma or dried blood spots. Inter-day precision was within 20% at each concentration of isobutyrylcarnitine and butyrylcarnitine. Precision for 2-methylbutyrylcarnitine and isovalerylcarnitine at concentrations above the normal range was within 24%. CONCLUSIONS: Two diagnostic tests based on the separation of C4-acylcarnitine and C5-acylcarnitine isomers by UPLC-MS/MS provide fast differential diagnosis of SCAD deficiency versus IBCD deficiency and IVA versus 2-MBCD deficiency. The separation of C5-acylcarnitines can reveal false elevation due to pivalic acid-containing antibiotics. Abnormal newborn screen results due to pivalate-generating prodrug antibiotics of maternal origin were confirmed. This separation of isomers can resolve multiple diagnostic challenges in both newborn screening and in cases with ambiguous metabolic test results.

Expanding metabolic screening of newborns: can the health care industry do better than public health?

In Oklahoma since the early 1990s, all newborns have been screened for four metabolic conditions: phenylketonuria, hypothyroidism, galactosemia and hemoglobinopathies. In 2002, 38 affected babies were diagnosed and one expects they are saved from the complications of late diagnosis such as mental retardation or death from sepsis. With advances in genetics and improved biochemical assays, 86% of states now screen for more disorders than Oklahoma, up to 37 in Mississippi. Six recent patient vignettes illustrate the mortality and morbidity of conditions that are screened for elsewhere but not in Oklahoma. In 2001, the Oklahoma Genetics Advisory Council recommended adding three disorders and the State Health Department forecasts that implementation may be complete in 2007. For now, when a patient asks, "Will my baby be screened for as many metabolic conditions as possible?", two answers represent either the public health or the private health care view. The public health answer is, "The state requires screening for four conditions." The health care system answer is, "We can work with you to get 44 conditions tested for, but it will cost money, may not be reimbursed, and has not been proven effective when done on an individual basis." This dilemma, not unique to newborn screening, might be resolved if professional and public opinion strongly supported early expansion.

Folic acid awareness and use among women with a history of a neural tube defect pregnancy--Texas, 2000-2001.

The use of folic acid is a critical component in preventing birth defects. Health-care providers should take advantage of all health-care visits to counsel not only women at high risk (i.e., those with a history of having an infant with a neural tube defect [NTD]) but all women regarding the importance of folic acid use. A study conducted in Texas confirmed that white and Hispanic mothers were equally likely to recall receiving postpartum advice to use folic acid; however, Hispanic women were much less likely to use folic acid, compared with white women. This report covers data from May 2000 through November 2001. A study was conducted in Texas to determine whether women at high risk recall and follow recommendations to use folic acid. The study included 195 women at high risk and 223 control mothers who gave birth to infants without birth defects. These women participated in a telephone interview for a population-based case-control study of NTDs. Approximately 56.4% (110 of 195) of mothers who had infants affected by an NTD recalled receiving postpartum advice to use folic acid, compared with 25.6% (57 of 223) of control mothers (p < 0.01). Among nonpregnant case mothers, 54 (32.7%) of 165 reported regular use of supplements containing folic acid, and 53 (25.2%) of 210 nonpregnant control mothers reported this behavior (p = 0.11). Among case mothers, use of folic acid was significantly higher for whites (64.7%) versus Hispanics (16.5%) (p < 0.001); for women with some college education (57.1%) versus no college education (20.2%; p < 0.001); for women who were trying to get pregnant (66.7%) versus those using birth control (38.3%) or reporting using no contraceptive method (18.8%) (p = 0.001); and for women who reported receiving advice to use folic acid (40.9%) versus those who did not (22.2%; p = 0.01). Findings from this study support the need to implement NTD recurrence prevention activities in Texas. Data also identify a need for educational strategies in Texas that target Hispanic women at high risk, especially those who primarily speak Spanish. Further efforts should be made to determine why Hispanic women have low rates of folic acid use (e.g., the cost of vitamins and language and cultural barriers). On the basis of a review of research and current practice, recommendations developed by the Public Health Service include 1) women at risk for a recurrent NTD-affected pregnancy should take 0.4 mg of folic acidper day; and 2) if a woman at high risk is planning a pregnancy, she should consult her physician regarding taking the higher dose of 4.0 mg per day.