Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-15 (of 15 Records) |
Query Trace: Chace L[original query] |
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Frontline field epidemiology training programs as a strategy to improve disease surveillance and response
Andre AM , Lopez A , Perkins S , Lambert S , Chace L , Noudeke N , Fall A , Pedalino B . Emerg Infect Dis 2017 23 (13) S166-73 Since 1980, Field Epidemiology Training Programs (FETPs) have trained highly qualified field epidemiologists to work for ministries of health (MOH) around the world. However, the 2013-2015 Ebola epidemic in West Africa, which primarily affected Guinea, Liberia, and Sierra Leone, demonstrated a lack of field epidemiologists at the local levels. Trained epidemiologists at these levels could have detected the Ebola outbreak earlier. In 2015, the US Centers for Disease Control and Prevention (CDC) launched FETP-Frontline, a 3-month field training program targeting local MOH staff in 24 countries to augment local public health capacity. As of December 2016, FETP-Frontline has trained 1,354 graduates in 24 countries. FETP-Frontline enhances global health security by training local public health staff to improve surveillance quality in their jurisdictions, which can be a valuable strategy to strengthen the capacity of countries to more rapidly detect, respond to, and contain public health emergencies at the source. |
Technological journey from colorimetric to tandem mass spectrometric measurements in the diagnostic investigation for phenylketonuria
Chace DH , Hannon WH . J Inborn Errors Metab Screen 2016 4 Phenylalanine analysis for phenylketonuria (PKU) detection in newborn screening (NBS) was chosen as the model system to describe how advancements in laboratory technology improved laboratory performance. These advancements have made NBS programs better and have improved the health outcomes of the affected newborn through improvements in accurate early detection over the past 50 years. The most current state-of-the-art technology, tandem mass spectrometry (MS/MS), has proven that it is now the choice in almost all modern NBS facilities because it is a versatile instrument that continues to grow in its application not just for amino acid and acylcarnitine detection but for other metabolites and disorders such as lysosomal storage diseases and second-tier detection of some screen-positive results. The use of MS/MS will continue to expand, even with the anticipated introduction and expansion of molecular screening methods into NBS programs. Regarding technological advancements, the future of NBS will include even newer technologies and approaches that will enhance the detection and treatment of newborns affected by PKU and other inborn errors of metabolism. |
Heptadecanoylcarnitine (C17) a novel candidate biomarker for newborn screening of propionic and methylmalonic acidemias
Malvagia S , Haynes CA , Grisotto L , Ombrone D , Funghini S , Moretti E , McGreevy KS , Biggeri A , Guerrini R , Yahyaoui R , Garg U , Seeterlin M , Chace D , De Jesus VR , la Marca G . Clin Chim Acta 2015 450 342-8 BACKGROUND: 3-Hydroxypalmitoleoyl-carnitine (C16:1-OH) has recently been reported to be elevated in acylcarnitine profiles of patients with propionic acidemia (PA) or methylmalonic acidemia (MMA) during expanded newborn screening (NBS). High levels of C16:1-OH, combined with other hydroxylated long chain acylcarnitines are related to long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and trifunctional protein (TFP) deficiency. METHODS: The acylcarnitine profile of two LCHADD patients was evaluated using liquid chromatography-tandem mass spectrometric method. A specific retention time was determined for each hydroxylated long chain acylcarnitine. The same method was applied to some neonatal dried blood spots (DBS) from PA and MMA patients presenting abnormal C16:1-OH concentrations. RESULTS: The retention time of the peak corresponding to C16:1-OH in LCHADD patients differed from those in MMA and PA patients. Heptadecanoylcarnitine (C17) has been identified as the novel biomarker specific for PA and MMA patients through high resolution mass spectrometry (Orbitrap) experiments. We found that 21 out of 23 neonates (22 MMA, and 1PA) diagnosed through the Tuscany region NBS program exhibited significantly higher levels of C17 compared to controls. Twenty-three maternal deficiency (21 vitamin B12 deficiency, 1 homocystinuria and 1 gastrin deficiency) samples and 82 false positive for elevated propionylcarnitine (C3) were also analyzed. CONCLUSIONS: We have characterized a novel biomarker able to detect propionate disorders during expanded newborn screening (NBS). The use of this new biomarker may improve the analytical performances of NBS programs especially in laboratories where second tier tests are not performed. |
Clinical chemistry and dried blood spots: increasing laboratory utilization by improved understanding of quantitative challenges
Chace DH , De Jesús VR , Spitzer AR . Bioanalysis 2014 6 (21) 2791-4 To best understand the use of dried blood spots (DBS) in clinical chemistry, we must first differentiate laboratory screening from diagnostics. Newborn screening (NBS) utilizes DBS to detect and quantify biomarkers indicative of more than 50 congenital diseases primarily of metabolic origin [1]. Many different methods are used, including modern analytical technologies such as tandem mass spectrometry (MS/MS) [2, 3] and molecular analyses [4, 5]. Each method is designed specifically to analyze DBS specimens with laboratory protocols and systems that begin with a paper hole puncher rather than a pipette. Unfortunately, NBS is often differentiated from clinical chemistry because it is “screening” tool rather than a “diagnostic” application. This is a common misconception because no method is diagnostic, but rather leads to a physician diagnosis that is based, in part, on laboratory results. Like any clinical test, NBS results take into consideration additional data such as age of newborn, birth weight, gestational age, and nutritional status [6–8]. Additionally, NBS is regulated by a comprehensive quality assurance/quality control (QA/QC) network that is shared worldwide [9]. There are a small number of traditional clinical laboratories that use DBS in a diagnostic setting, primarily in specialized metabolic applications, further supporting that DBS utilization is not restricted to “screening” laboratories, nor does it involve relaxed laboratory standards. The question is: why are DBS not used to a greater extent in traditional clinical laboratories? It is likely due to the analytical challenges and common misperceptions of the DBS. Specifically, the challenges of DBS implementation such as cross-validation of a DBS assay from a liquid sample analysis, logistical systems limitations for handling DBS versus liquid samples, and clinical laboratory bias towards single metabolite analysis versus metabolic profiles. |
Metabolomic profiling of amino acids and beta-cell function relative to insulin sensitivity in youth
Michaliszyn SF , Sjaarda LA , Mihalik SJ , Lee S , Bacha F , Chace DH , De Jesus VR , Vockley J , Arslanian SA . J Clin Endocrinol Metab 2012 97 (11) E2119-24 CONTEXT: In longitudinal studies of adults, elevated amino acid (AA) concentrations predicted future type 2 diabetes mellitus (T2DM). OBJECTIVE: The aim of the present investigation was to examine whether increased plasma AA concentrations are associated with impaired beta-cell function relative to insulin sensitivity [i.e. disposition index (DI)], a predictor of T2DM development. DESIGN, SETTING, AND PARTICIPANTS: Metabolomic analysis for fasting plasma AAs was performed by tandem mass spectrometry in 139 normal-weight and obese adolescents with and without dysglycemia. First-phase insulin secretion was evaluated by a hyperglycemic ( approximately 225 mg/dl) clamp and insulin sensitivity by a hyperinsulinemic-euglycemic clamp. DI was calculated as the product of first-phase insulin and insulin sensitivity. RESULTS: DI was positively associated with branched-chain AAs (leucine/isoleucine and valine; r = 0.27 and 0.29, P = 0.001), neutrally transported AAs (phenylalanine and methionine; r = 0.30 and 0.35, P < 0.001), basic AAs (histidine and arginine; r = 0.28 and 0.23, P ≤ 0.007), serine (r = 0.35, P < 0.001), glycine (r = 0.26, P = 0.002), and branched-chain AAs-derived intermediates C3, C4, and C5 acylcarnitine (range r = 0.18-0.19, P ≤ 0.04). CONCLUSION: In youth, increased plasma AA concentrations are not associated with a heightened metabolic risk profile for T2DM; rather, they are positively associated with beta-cell function relative to insulin sensitivity. These contrasting observations between adults and youth may be a reflection of developmental differences along the lifespan dependent on the combined impact of the aging process together with the impact of progressive obesity. |
Metabolomic profiling of fatty acid and amino acid metabolism in youth with obesity and type 2 diabetes: evidence for enhanced mitochondrial oxidation
Mihalik SJ , Michaliszyn SF , de las Heras J , Bacha F , Lee S , Chace DH , DeJesus VR , Vockley J , Arslanian SA . Diabetes Care 2012 35 (3) 605-11 OBJECTIVE: We compared acylcarnitine (AcylCN) species, common amino acid and fat oxidation (FOX) byproducts, and plasma amino acids in normal weight (NW; n = 39), obese (OB; n = 64), and type 2 diabetic (n = 17) adolescents. RESEARCH DESIGN AND METHODS: Fasting plasma was analyzed by tandem mass spectrometry, body composition by dual energy X-ray absorptiometry and computed tomography, and total-body lipolysis and substrate oxidation by [(2)H(5)]glycerol and indirect calorimetry, respectively. In vivo insulin sensitivity (IS) was assessed with a 3-h hyperinsulinemic-euglycemic clamp. RESULTS: Long-chain AcylCNs (C18:2-CN to C14:0-CN) were similar among the three groups. Medium- to short-chain AcylCNs (except C8 and C10) were significantly lower in type 2 diabetes compared with NW, and when compared with OB, C2-, C6-, and C10-CN were lower. Amino acid concentrations were lower in type 2 diabetes compared with NW. Fasting lipolysis and FOX were higher in OB and type 2 diabetes compared with NW, and the negative association of FOX to C10:1 disappeared after controlling for adiposity, Tanner stage, and sex. IS was lower in OB and type 2 diabetes with positive associations between IS and arginine, histidine, and serine after adjusting for adiposity, Tanner stage, and sex. CONCLUSIONS: These metabolomics results, together with the increased rates of in vivo FOX, are not supportive of defective fatty acid or amino acid metabolism in obesity and type 2 diabetes in youth. Such observations are consistent with early adaptive metabolic plasticity in youth, which over time-with continued obesity and aging-may become dysfunctional, as observed in adults. |
Evaluation of asymmetric dimethylarginine, arginine, and carnitine metabolism in pediatric sepsis
Weiss SL , Haymond S , Ranaivo HR , Wang D , De Jesus VR , Chace DH , Wainwright MS . Pediatr Crit Care Med 2012 13 (4) e210-8 OBJECTIVE: Increased plasma concentrations of the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine, decreased arginine bioavailability, and mitochondrial dysfunction have been reported in adult sepsis. We studied whether asymmetric dimethylarginine, arginine, and carnitine metabolism (a measure of mitochondrial dysfunction) are altered in pediatric sepsis and whether these are clinically useful biomarkers. DESIGN: Prospective, observational study. SETTING: Pediatric intensive care unit at an academic medical center. PATIENTS: Ninety patients ≤18 yrs old, 30 with severe sepsis or septic shock, compared with 30 age-matched febrile and 30 age-matched healthy control subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma asymmetric dimethylarginine and whole blood arginine, citrulline, ornithine, and acylcarnitine:free carnitine ratio were measured daily for septic patients and once for control subjects using tandem mass spectrometry. Plasma asymmetric dimethylarginine concentration (median; interquartile range mumol/L) on day 1 was lower in severe sepsis and septic shock (0.38; 0.30-0.56) compared with febrile (0.45; 0.40-0.59) and healthy (0.60; 0.54-0.67) control subjects (p < .001), although decreased asymmetric dimethylarginine was predominantly found in neutropenic patients. Day 1 arginine was lower in septic (10; interquartile range, 7-20 mumol/L) compared with healthy patients (32; interquartile range, 23-40; p < .001), and the arginine:ornithine ratio was decreased in sepsis, indicating increased arginase activity (an alternative pathway for arginine metabolism). The arginine:asymmetric dimethylarginine and acylcarnitine:free carnitine ratios did not differ between septic and control patients. Asymmetric dimethylarginine was inversely correlated with organ dysfunction by Pediatric Logistic Organ Dysfunction score (r = -0.50, p = .009), interleukin-6 (r = -0.55, p = .01), and interleukin-8 (r = -0.52, p = .03) on admission. Arginine, arginine:asymmetric dimethylarginine, and acylcarnitine:free carnitine were not associated with organ dysfunction or outcomes. CONCLUSIONS: Asymmetric dimethylarginine was decreased in pediatric sepsis and was inversely associated with inflammation and organ dysfunction. This suggests that inhibition of nitric oxide synthase by asymmetric dimethylarginine accumulation is unlikely to impact sepsis pathophysiology in septic children despite decreased arginine bioavailability. We did not find an association of asymmetric dimethylarginine with altered carnitine metabolism nor were asymmetric dimethylarginine, arginine, and acylcarnitine:free carnitine useful as clinical biomarkers. |
Commentary on the history and quantitative nature of filter paper used in blood collection devices
De Jesus VR , Chace DH . Bioanalysis 2012 4 (6) 645-7 It is with great interest that we read Peter T. Kissinger’s commentary about the use of dried-blood spots (DBS) for pharmacokinetic assays and therapeutic drug monitoring (Bioanalysis 2011, 3(20), 2263–2266). We appreciate Dr. Kissinger’s efforts to stimulate debate regarding the utility of DBS in these two types of applications. However, we would like to dispel some misconceptions about DBS and their filter paper matrix that readers, unaware of their many complexities, may derive from his commentary. Our intent is to provide information supporting the use of DBS in many applications, based on 50 years of DBS use by newborn screening (NBS) laboratories in the US. |
Detection of TPN contamination of dried blood spots used in newborn and metabolic screening and its impact on quantitative measurement of amino acids
Chace DH , De Jesus VR , Lim TH , Hannon WH , Clark RH , Spitzer AR . Clin Chim Acta 2011 412 1385-90 BACKGROUND: Markers derived from dextrose (d-glucose) are observed in the MS/MS-based acylcarnitine profiles from dried-blood spots of some premature infants receiving intravenous nutrition. The presence of these markers at m/z 325, 399 and 473 are thought to arise from contamination of blood by total parenteral nutrition (TPN) solutions during specimen collection from premature infants. These solutions contain high concentrations of amino acids and as a result, false-positive screening results for amino acid disorders may occur. This study investigates quantitative parameters of dextrose and amino acids in blood samples enriched with different TPN solutions. METHODS: Whole blood collected in heparin was enriched with three different TPN solutions containing 5, 10 or 12.5% dextrose and amino acids that were originally prepared for delivery of 2.5, 3 or 4g/kg/day of Premasol(R) then spotted onto filter paper cards. Acylcarnitine and amino acid profiles using MS/MS were obtained. Ion ratios of dextrose relative to specific acylcarnitine stable isotope internal standards and amino acid concentrations were obtained. RESULTS: The ion ratios for each of the dextrose markers at m/z 325, 399 and 473 exhibit linearity with the concentration of the dextrose component of TPN added to blood. The lowest detectable dextrose concentration added to blood was 7.6mmol/l at 1:80v/v TPN in blood. Furthermore, the concentrations of amino acids were linear with the concentration of the amino acid component of TPN added to blood. At the lowest detectable concentrations of dextrose marker, the amino acid concentrations were at or above the values considered abnormal in newborn screening laboratories. The molar ratios of amino acids approached the relative quantity of amino acid in the TPN solution with increasing enrichments in blood. CONCLUSIONS: Detection of the combinations of dextrose markers, very high elevations of amino acids and unusual molar ratios can be used to reject a specimen as improperly collected rather than declaring it a false positive and hence reduce false positive rates. This process enhances efficiency, reduces parental anxiety, and improves positive predictive values. |
Proficiency testing outcomes of 3-hydroxyisovalerylcarnitine measurements by tandem mass spectrometry in newborn screening
Lim TH , De Jesus VR , Meredith NK , Sternberg MR , Chace DH , Mei JV , Hannon WH . Clin Chim Acta 2010 412 631-5 BACKGROUND: The use of tandem mass spectrometry (MS/MS) for the analysis of amino acids and acylcarnitines from dried-blood spots (DBS) has become routine practice in newborn screening laboratories. The Newborn Screening Quality Assurance Program (NSQAP) added 3-hydroxyisovalerylcarnitine (C5OH) into its routine quality control and proficiency testing (PT) DBS materials for MS/MS to assure the quality of C5OH screening. We report the results from NSQAP evaluations for C5OH-enriched DBS, and summarize participant screening practices based on their analytical methods. METHODS: NSQAP prepared C5OH-enriched DBS materials for its participants. Laboratories reported quantitative and qualitative results. Bias plots of quantitative results were constructed using reported data and the results were sorted by analytical method. RESULTS: NSQAP participants reported PT specimen 3964 as outside of normal limits for C5OH. The mean C5OH value for derivatized and non-derivatized methods was 2.80 and 2.67mumol/l, respectively. Reported data from other specimens showed a similar trend in derivatized vs. non-derivatized assay results. Differences in C5OH quantitative values were observed among laboratories using different internal standards. CONCLUSIONS: C5OH MS/MS measurements in DBS assays varied by method and the choice of internal standards. The use of NSQAP's DBS materials allows harmonization of C5OH measurements by newborn screening laboratories worldwide. |
Impact of second-tier testing on the effectiveness of newborn screening
Chace DH , Hannon WH . Clin Chem 2010 56 (11) 1653-5 The goal of newborn screening (NBS)3 for inherited disorders of metabolism is the early detection and confirmation of disease, thus enabling early medical intervention, treatment, and improved outcomes (1). Important characteristics of a screening method include analytical specificity and sensitivity, coupled with rapid, high throughput and timely reporting of abnormal results. Routine primary screening methods are designed to identify as many abnormal infants as possible, with diagnostic sensitivity favored over specificity for disorder detection. This approach not only increases the numbers of false-positive test results, thus adding to the cost of operating NBS programs, but also places unnecessarily increased stress, anxiety, and possibly parent–child dysfunction on families (2). As the number of disorders in the NBS test panels grows, however, so does the overall number of false-positive results, which has increased severalfold per true case (3). One solution to this problem is to use improved methods or to couple primary screening methods with second-tier tests that improve selectivity. | The use of tandem mass spectrometry (MS/MS) for detecting phenylketonuria is an example of an NBS method that improves detection as a primary screen while also being more selective than older, classic NBS methods such as fluorometry. In one study, MS/MS analysis of newborn spot samples of dried blood collected ≤24 h after birth was compared with fluorometric analysis of the same samples. Because of this early time of collection, the decision level for an increased phenylalanine concentration was lowered by the public health laboratory using fluorometry to ensure that no infants with phenylketonuria were missed. MS/MS analysis of the identical samples demonstrated that disease detection could be sustained while improving selectivity (4). The ability to measure multiple analytes in the same analysis enabled the calculation of the phenylalanine/tyrosine molar ratio, which reduced false-positive rates a 100-fold. This screen for phenylketonuria was the first instance of a new paradigm in NBS, in which both current screens could be improved and new screens could be added for other disorders, such as fatty acid oxidation defects and organic acidemias (5). The ability of MS/MS to improve efficacy without the need for collecting a second sample reduces the false-positive rate. |
Tandem mass spectrometric identification of dextrose markers in dried-blood spots from infants receiving total parenteral nutrition
Chace DH , De Jesus VR , Lim TH , Hannon WH , Spitzer AR . Clin Chim Acta 2010 411 1806-16 BACKGROUND: The false positive rate for the newborn screening of disorders of amino acid metabolism for premature infants is higher than full term infants. This may be due to very low birth weight infants receiving high concentrations of amino acids from total parenteral nutrition (TPN) administration and/or immature metabolism. An investigation of the possible influence of TPN on screening of premature infants resulted in the detection of three unusual peaks in the tandem mass spectrometry (MS/MS) acylcarnitine profile. These markers were closely correlated with the detection of very high multiple amino acid increases in the profiles of newborns administered with TPN and who were ultimately found to be normal and free of inherited metabolic disorders. METHODS: TPN solutions contain a concentrated mixture of amino acids and dextrose and other nutrients in saline. Due to its high concentration and suggestion of a carbohydrate, it was hypothesized that dextrose (D-glucose) was the contaminant and source of the markers detected. Dextrose, stable isotope-labeled 13C6-dextrose and various TPN solutions were analyzed directly or after enrichment in whole blood by multiple MS/MS acquisition modes including MS-only, product and precursor ion and neutral loss scans. RESULTS: Analysis of dried-blood spots (DBS) prepared from whole blood spiked with TPN solutions containing 12.5% dextrose and amino acid formulations designed to deliver 2.5 gm/kg/day of an amino acid mixture had moderate increases of all 3 dextrose markers detected at m/z 325, 399 and 473 as compared to controls. MS-only scans, product and precursor ion scans of dextrose and 13C6-dextrose in positive ion mode confirmed that these 3 peaks are derived from dextrose. Mass spectral analysis of labeled and unlabeled dextrose suggested that these peaks were dimers derived from dextrose. CONCLUSION: The identification of dextrose markers in DBS indicates that high concentrations of dextrose were present in blood and the likely source was contamination by TPN solutions most likely occurring during a sample collection process. |
Potential loss of methionine following extended storage of newborn screening samples prepared for tandem mass spectrometry analysis
Chace DH , Luo Z , De Jesus VR , Haynes CA , Hannon WH . Clin Chim Acta 2010 411 1284-6 BACKGROUND: Methionine (Met) is a key metabolite used in the newborn screening of homocystinuria by tandem mass spectrometry (MS/MS). Recently, a loss of ion counts in both Met and its deuterium-labeled internal standard ((2)H(3)-Met) was observed by the CDC's Newborn Screening Quality Assurance Program laboratory. We report on the stability of labeled and unlabeled Met solutions and their storage in two types of 96 well microtiter plates to illustrate the potential loss of Met following storage of samples prior to MS/MS analysis. METHODS: Neat labeled and unlabeled Met standards were prepared and added (25, 50 and 100 microl) to two different types of microtiter plates, dried under nitrogen and stored for up to 168 h. All samples were reconstituted in mobile phase and analyzed as free acids for simplification of the study. RESULTS AND CONCLUSIONS: Met appears to interact significantly with polystyrene microtiter plates and to a much lesser extent with polypropylene microtiter plates. Furthermore, the loss is greatest for lower concentrations of methionine. While this loss of Met signal may be unimportant due to a presumption of equal loss of (2)H(3)-Met, a significant decline in ion signals will cause greater error in the calculation of concentration. These results suggest that polypropylene may be a better choice for Met analysis. Furthermore, storing prepared samples prior to analysis may impact the quality of the MS/MS analysis for Met and potentially other metabolites. Plates used by newborn screening laboratories should be evaluated periodically if the signal intensity for Met is reduced. |
Comparison of amino acids and acylcarnitines assay methods used in newborn screening assays by tandem mass spectrometry
De Jesus VR , Chace DH , Lim TH , Mei JV , Hannon WH . Clin Chim Acta 2010 411 684-9 BACKGROUND: The analysis of amino acids (AA) and acylcarnitines (AC) by tandem mass spectrometry (MS/MS) is performed in newborn screening laboratories worldwide. While butyl esterification assays are routine, it is possible to detect AAs and ACs as their native free acids (underivatized). The Centers for Disease Control and Prevention's Newborn Screening Quality Assurance Program provides dried-blood spot (DBS) quality control (QC) and proficiency testing (PT) programs for numerous MS/MS analytes. We describe empirical differences between derivatization and non-derivatization techniques for selected AAs and ACs. METHODS: DBS materials were prepared at levels near, above and below mean domestic laboratory cut-offs, and distributed to program participants for MS/MS analysis. Laboratories reported quantitative and qualitative results. QC DBS materials were assayed in-house following established protocols. RESULT: Minor differences (<15%) between quantitative values resulting from butyl esters and free acid techniques were observed for the majority of the analytes. Mass spectrometric response from underivatized dicarboxylic acid acylcarnitines was less intense than their butyl esters. CONCLUSIONS: The use of underivatized techniques may also result in the inability to differentiate isobaric acylcarnitines. Laboratories should establish their own protocols by focusing on the decisions that identify test results requiring additional follow-up testing versus those that do not. |
Improved MS/MS analysis of succinylacetone extracted from dried blood spots when combined with amino acids and acylcarnitine butyl esters
Chace DH , Lim T , Hansen CR , De Jesus VR , Hannon WH . Clin Chim Acta 2009 407 6-9 BACKGROUND: The utilization of succinylacetone (SUAC) as the primary metabolic marker for tyrosinemia Type I is now well known, thus new methods have been developed to analyze SUAC as a first tier test in newborn screening. One approach is to prepare a SUAC hydrazine derivative from the dried blood spots (DBS) previously utilized in the extraction of acylcarnitine (AC) and amino acids (AA). The final derivatized products of SUAC, AA and AC are combined in a single tandem mass spectrometric (MS/MS) analysis. However, butyl esterification techniques may result in contamination of underivatized acylcarnitines by as much as 20%. We have developed a simple wash step to improve the combined analysis of SUAC, AA and AC in DBS by MS/MS. METHODS: AA and AC were extracted with methanol containing labeled internal standard from 3.2mm punches taken from the DBS specimen. The previously extracted blood spot that remains after removal of the methanol extraction solvent was used in the preparation of SUAC with and without additional washing of the blood spot. The butyl ester eluates of AA and AC, and SUAC hydrazine derivatives were recombined and measured by MS/MS. RESULTS: Three additional methanol wash steps of the remaining DBS punches prior to SUAC derivatization reduced the presence of underivatized acylcarnitines, resulting in a 4-fold reduction of underivatized palmitoylcarnitine. Palmitoylcarnitine butyl ester is detected at m/z 456 while the underivatized species is detected at m/z 400, which is also the mass of dodecanoylcarnitine butyl ester. The linearity of the SUAC assay was unchanged by the additional wash steps. For butyl esterification methods, the preferred analytic procedure, the presence of AC can compromise the results of a newborn screen for the actual concentrations of acylcarnitines. It is essential to remove any underivatized acylcarnitines prior to SUAC analysis. CONCLUSION: The additional methanol wash steps did not alter SUAC assay results but did remove underivatized acylcarnitines which could result in the incorrect quantification of acylcarnitines. |
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